CA2266010C - Thermal cycler for automatic performance of the polymerase chain reaction with close temperature control - Google Patents

Abstract

An instrument for performing highly accurate PCR
employing a sample block in microtiter tray format. The sample block has local balance and local symmetry. A three zone film heater controlled by a computer and ramp cooling solenoid valves also controlled by the computer for gating coolant flow through the block controls the block temperature. Constant bias cooling is used for small changes. Sample temperature is calculated instead of measured. A heated cover deforms plastic caps to apply a minimum acceptable threshold force for seating the tubes and thermally isolates them. The control software includes diagnostics. An install program tests and characterizes the instrument. A new user interface is used. Disposable, multipiece plastic microtiter trays to give individual freedom to sample tubes are taught.

Description

T~MA~ CY~TF~ FOR AUTOMATIC P~RFORMANC~ OF THE
POTYM~ASE ~IN ~F~CTION WITH ~T~S~ TF~P~ATURF- CONTROT
The present application is a divisional application of Canadian Patent Application Serial No 2,056,743, 5 filed November 29, 1991 Backqround of the Invention The invention pertain~ to the fi-ld of computer directed instruments for p-rforming the polymerase chain 10 reaction (hereafter PCR) More particularly, the invention pertains to automated in~trum-nt~ that can perform the polymerase chain reaction cimultan-ously on many samples with a very high degree of pr-cision as to results obtained for each sample This high precision provides the 15 capability, among other things, of performing so-called "guantitative PCR"
To amplify DNA (Deoxyribo~e Nucleic Acid) using the PCR
process, it is necessary to cycle a specially constituted liquid reaction mixture through a PCR protocol including 20 several different temperature incubation periods The reaction mixture i- compri~-d of various component~ such as the DNA to be amplified and at least two primers selected in a predetermined way so as to be sufficiently complementary to the cample DNA as to be able to create exten6ion products 25 of the DNA to be amplified The r-action mixture includes various enzymes and/or other re~gents, as well as several deoxyribonucleoside triphosphat-- such as dATP, dCTP, dGTP
and dTTP Generally, the primers ar- oligonucleotide~ which are capable of acting as a point of initiation of synthesis 30 when placed under conditions in which synthesi6 of a primer extension product which is complimentary to a nucleic acid strand is induced, i e , in the presence of nucl-otides and inducing agent6 ~uch as thermostable DNA polymerase at a suitable temperature and pH
The Polymerase Chain Reaction (PCR) has proven a phenomenally ~uccessful technology for genetic analysis, largely because it i- ~o ~impl- and reguire- relatively low cost instrumentation A key to PCR i6 the concept of thermocycling alternating ~t~ps of ~elting DNA, annealing 5 short primers to the resulting ~ingle ~trands, and extending those primers to make new copies of double stranded DNA In thermocycling, the PCR reaction mixture is repeatedly cycled from high temper~tures (~90~ C) for melting the DNA, to lower temperatures (40'C to 70 C) for prim-r annealing and 10 extension The first commercial ~y-t~m for performing the thermal cycling required in the polymerase chain reaction, the Perkin-Elmer Cetus DNA Thermal Cycler, wa- introduced in Applications of PCR technology are now moving from basic 15 research to applications in which large numbers of similar Amplifications are routinely run These areas include diagnostic research, biopharmaceutical development, genetic analysis, and environmental testing U~ers in these areas would benefit from a high perfor~anc- PCR system that would 20 provide the user with high throughput, rapid turn-around time, and reproducibl- results U-ers in these areas must be assured of reproducibility from sample-to-sample, run-to-run, lab-to-lab, and instrument-to-instrument For example, the physical ~apping process in the Human 25 Genome Project may become greatly ~implified by utilizing sequence tagg-d sites An STS i- a ~hort, uniqu- sequence easily amplified by-PCR and which identifi-~ a location on the chromo~ome Ch-c~ing for ~uch ~ites to mak- genome maps requires amplifying large numbers of ~amples in a ~hort time 30 with protocols which can b- r-producibly run throughout the world As the nu~ber of PCR samples increa~es, it becomes more important to integrate amplification with sample preparation and post-amplification analysis The sample ve-sels must 35 not only allow r~pid thermal cycling but also permit more autom~ted h~ndling for oper~tions ~uch as solvent , .

extractions and centrifuqation. The vessels ~hould work consistently at low volumes, to reduce reagent costs.
Generally PCR temperature cycling involves at least two incubations at different temperatures. One of these 5 incubations is for primer hybridization and a catalyzed primer extension reaction. The other incubation is for denaturation, i.e., ~eparation of the double stranded extension products into single strand template~ for use in the next hybridization and ~xtension ~nc~hation interval.
10 The details of the polymerase chain r-action, the temperature cycling and reaction conditions n~res~ary for PCR as well as the various reagents and enzymes necessary to perform the reaction are described in U.S. patents 4,683,202, 4,683,195, EPO Publication 258,017 and 4,889,818 15 (Tag polymerase enzyme patent) ~nd all other PCR patents which are assigned to Cetus Corporation.

The purpose of a polymerase chain reaction is to manufacture a large volume of DNA which i- identical to an 20 initially supplied small volume of ~seed" DNA. The reaction involves copying the strands of the DNA and then using the copies to generate other copies in subseguent cycles. Under ideal conditions, each cycle will double the amount of DNA
present thereby r-sulting in a geometric progression in the 25 volume of copies of the ~target~ or ~seed~ DNA strands present in th- reaction mixture.
A typical PCR temperature cycle requires that the reaction mixture be held accurat-ly at ~ach incubation temperature for a prescribed time and that the identical 30 cycle or a similar cycle be repeated many times. A typical PCR progran starts at a sample temperature of 94-C held for 30 seconds to denature t~e r-action mixture. Then, the temperature of the reaction rixture is lowered to 37-C and held for one minute to permit primer hybridization. Next, 35 the temperature of the reaction mixture is raised to a temperture in the range from 50-C to 72-C w~ere it is ~eld for two minutes to promote the ~ynthesis of extension products This completes one cycle The next PCR cycle then starts by raising the temperature of thc reaction mixture to 94~C again for strand ~eparation of the extension 5 products formed in the previous cycle (denaturation) Typically, the cycle i- repeated 25 to 30 times Generally, it is desirable to change the sample temperature to the next temperature in the cycle as rapidly as possible for several rea~ons First, the chemical 10 reaction has an optimum temperature for each of it~ ~tages Thus, less time spent at nonoptimum temperatures means a better chemical result is achieved Another reason is that a minimum time for holding the reaction mixture at each incubation temperature i- reguired after each said 15 incubation temperature is r-ach-d Th-se minimum incubation times establish the ~floor~ or minimum time it takes to complete a cycle Any time transitioning betw-en sample incubation temperatures i- time which is added to this minimum cycle ti~e Since the number of cycles is fairly 20 large, this additional time unnecessarily lengthens the total time n-eded to compl-te the amplification In ~ome prior automat-d PCR in-trument-, th- reaction mixture was stored in a dib~c~hle plastic tube which is closed with a cap A typical ~ample volume for ~uch tubes 25 was approximat-ly 100 microliters Typically, ~uch instruments u-ed many ~uch tube~ filled with ~ample DNA and reaction mixture inserted into holes call-d ~ample wells in a metal block To perform the PCR p~o_~s-, the temperature of the metal block was controlled according to prescribed 30 temperatures and times ~pecified by the u-er in a PCR
protocol file A computer and associat-d electronics then controlled the temperature of th- metal block in accordance with the user ~uppli-d data in the PCR protocol file defining the times, temperatures and numb-r o~ cycles, etc 35 As the metal block changed temperature, the samples in the various tubes followed with similar changes in temperature However, in these prior art instruments not all samples experienced exactly the ~ame temperature cycle In these prior art PCR instruments, errors in sample temperature were generated by nonuniformity of temperature from place to 5 place within the metal sample block, i e , temperature gradients existed within the metal of the block thereby causing some samples to have different temperatures than other samples at particular times in the cycle Further, there were delays in trancferring heat from the aample block 10 to the sample, but the delays were not the ~ame for all samples To perform the PCR process succes~fully and efficiently, and to enable so call-d "quantitative" PCR, these time delays and temperature errors must be minimized to a great extent The problems of minimizing time delayc for heat transfer to and from the sample liguid and minimizing temperature errors due to temperature gradients or nonuniformity in temperature at various points on the metal block become particularly acute when the size of the region 20 containing samples becomes large It is a highly desirable attribute for a PCR in-trument to have a metal block which is large enough to accommodatc 96 sample tubes arranged in the format of an industry ~tandard microtiter plate The microtiter plate is a widely used means for 25 handling, processing ~nd analyzing larg- number- of small samples in the biochemistry and biot-chnology fields Typically, a micr~titer plate is a tray which is 3 5/8 inches wide and 5 inches long and contains 96 identical sample wells in an 8 well by 12 well rectangular array on 9 30 millimeter centers Although microtiter plates are available in a wide variety of materials, shapes and volumes of the sample wells, which are optimized for many different uses, all microtiter plat-s have the same ovcrall out ide dimensions and the same 8 x 12 array of wells on 9 35 millimeter centers A wide variety of eguipment is available for automating t~e handling, processing and analyzing of samples in this standard microtiter plate format Generally microtiter plates are made of injection molded or vacuum formed plastic and are inexpensive and 5 considered disposable Disposability is a highly desirable characteristic because of the legal liability arising out of cross contamination and the difficulty of washing and drying microtiter plates ~fter use It is therefore a highly desirable characteristic for 10 a PCR instrument to be able to perform the PCR reaction on up to 96 samples simultaneously said s~mples being arranged in a microtiter plate format Of course, the si2e of the metal block which is necessary to heat and cool 96 ~amples in an 8 x 12 well 15 array on 9 millimeter centers i- fairly large This large area block creates multiple challenging ~ngineering problems for the design of a PCR instrument which is capable of heating and cooling such a block very rapidly in a temperature range generally from 0 to lOO C with very little 20 tolerance for temperature variations between samples These problems arise from sevQral ~ourc-s First, the large thermil mass of the block makes it difficult to move the block temperature up and down in the operating range with great rapidity Second, the need to attach the block to 25 various external d-vices ~uch as manifolds for supply and withdrawal of cooling liquid, block cupport attachment points, and ~ssocia-ted other peripheral equipment creates the potential for temperature gradients to ~xist across the block which exceed tolerable limits There are alco numerou- other conflicts bet~een the requirements in the design of a thermal cycling system for automated performance of the PCR reaction or other reactions requiring rapid, accurate temperature cycling of a large number of samples For example, to change the temperature 35 of a metal block rapidly, a large amount of heat must be added to, or removed from the samplc block in a short period of time Heat can be added from electrical resistance heaters or by flowing a heated fluid in contact with the block Heat can be removed rapidly by flowing a chilled fluid in contact with the block However, it i- ~eemingly 5 impossible to add or remove large amounts of heat rapidly in a metal block by these means without causing large differences in temperature from place to place in the block thereby forming temperature gradient~ which can result in nonuniformity of temperature among the~samples Even after the process of addition or removal of heat is terminated, temperature gradient6 can per~i~t for a time roughly proportional to the ~quare of the di-tance that the heat stored in various points in the block must travel to cooler regions to eliminate the t mp-rature gradi-nt Thus, 15 as a metal block i- made larger to accommodate more ~amples, the time it takes for temperature gradients existing in the block to decay after a temperat-- change causes temperature gradients which extend across the largest dimension6 of the block can become mark~dly longer Thi6 makes it 20 increasingly difficult to cycle the temperature of the sample block rapidly while maintaining accurate temperature uniformity among all the samples Because of the time requir-d for temperature gradients to dissipate, an important need has ari~en in the design of 25 a high performance PCR instrument to prevent the creation of temperature gradient~ that extend over large distances in the block Anothe~ need i~ to avoid, a6 much a~ possible, the requirement for heat to travel acros~ mechanical boundarie~ between metal part~ or other p-ripheral ~quipment 30 attached to the block It i~ difficult to ~oin metal parts in a way that insures uniformly high thermal conductance everywhere across the joint Nonuniformitie6 of thermal conductance will generate unwanted temperature gradients summarY of the Invention According to the teachings of the inv-ntion, there is _ disclosed herein a thin walled eample tube for decreasing the delay between changes in ~ample temperature of the sample block and corresponding changes in temperature of the reaction mixture Two different ~ample tube sizes are 5 disclosed, but each has a thin walled conical ~ection t~hat fits into a matching conical recess in the sample block Typically, cones with 17~ angles relative to the longitudinal axis are used to prevent jamming of the tubes into the sample block but to allow snug fit Other shapes 10 and angles would also ~uffice for purpo~es of practicing the invention Also, other types of heat exch~nger~ can also be used other than sample blocks ~uch as liguid bath6, ovens, etc However, the wall thickness of the section of the ~ample 15 tube which is in contact with whatever heat exchange is being used should be as tmln as possible ~o long as it is sufficiently strong to withstand the thermal ~tresses of PCR
cycling and the stresses of norm~l u8e. Typically, the sample tubes are made of autoclavable polypropylene such as 20 Himont PD701 with a wall thickne~s of the conical ~ection in the range from 0 009 to 0 012 inche- plus or minus 0 001 inches Most preferably, the wall thickne~s is 0 012 inches In the preferred embodiment, the ~ample tube also has 25 a thicker walled cylindrical s-ction which joins with the conical ~ection This conical section provide containment for the original reaction miYture or reagents which may be added after PCR ~L C ~e eeing The ~ample tube ~hown in Figure 50 bas industry 30 standard configuration -Y-e~L ~or tb- thin wall~ for compatibility in otber PCR ~ystem~ The ~ample tube of Figure 15 is a shorter tube which can be used with the system disclosed herein The 35 other subject matter of the system en~ironment in which use of the thin walled sample tubes is preferred are ~ummarized * Trade-mark below There is also described h-rein a novel method and apparatus for achieving very accurate temp-rature control for a very large number of samples arranged in the 5 microtiter plate format during the performance of very rapid te3perature cycling PCR protocols The teachings of the invention contemplate a novel structure for a sample block, sample tubes and supporting mounting, heating and cooling apparatus, control electronics and software, a nov~l user 10 interface and a novel method of using ~aid apparatus to perform the PC~ protocol The instrument described herein is designed to do PCR
gene amplification on up to 96 ~amples with very tight tolerances of temperature control across the universe of 15 samples This mean~ that all samples go up and down in temperature simultaneously with very little difference in temperature between different wells containing different samples, this being true throughout the polymerase chain reaction cycle The instrument d-ccrib-d her-in is also 20 capable of very tight control of the reaction mixture concentration through control of the ~vaporation and condensation processes in ~ach ~ample well Further, the instrument described herein i6 capable of processing up to 96 samples of 100 ~icroliters each from different donor 25 sources with substantially no crosc-contamination between sample wells The teachings of th- invention h-rein includes a novel method of heating and cooling an aluoinum sample block to thernally cycl- samples in the st~n~rd 96-w-ll microtiter 30 plate format with the result that excellent sample-to-sample uniformity exists despite rapid thermal cycling rates, noncontrolled varying ambient temperatures and variations in other operating conditions such as power line voltage and coolant temperatures The tcachings of the invention also contemplate a novel design for a disposable plastic 96-well microtiter plate for . _ . .

accommodation of up to 96 individual ~ample tubes containing DNA for thermal cycling each eample tube having individual freedom of movement sufficient to find the best fit with the sample block under downward pressure from a heated cover 5 The microtiter plate design, by allowing each tube to find the best fit, provides high and uniform thermal conductance from the sample block to each sample tube even if differing rates of thermal expansion and contraction between the me~al of the block and the plastic of the aa~ple tube and 10 microtiter plate structure cau-e the relative center-to-center dimensions of the wells in the ~ampl- block to change relative to the center-to-center distance of the sample tubes in the disposable microtiter plate structure The teachings of the invention also contemplate a novel 15 method and apparatus for controlling the PCR instrument which includes the ability to continuously calculate and display the temperature of the ~amples being processed without directly measuring these temperatures These calculated temperatures are used to control the time that 20 the samples are held within the given temperature tolerance band for each targ-t temperature of incubation The control system also controls a three-zone heater thermally coupled to the sample block and gates fluid flow through directionally interlac-d ramp cooling channel~ in the sample 25 block which, when combined vith a constant bia- cooling flow of coolant through the ~ample block provides a facility to achieve rapid temperature changes to and pr-ci-- temperature control at targ-t temperatures specifi-d by the user The method and apparatus for controlling the thr-e-zone heater 30 includes an apparatus for taking into account, among other things, the line voltage, block temperature, coolant temrerature and ambient temperature in calculating the amount of electrical energy to be supplied to the various zones of the three-zone heater This heater has zones which 35 are separately controllable under the edges or "guard bands"
of the sample block so that excess heat losses to the ambient through peripheral ~guipment attached to the ~dges of the sample block can be compensated This helps prevent thermal gradients from forming The teachings of the invention al60 contemplate a novel 5 method and apparatus for preventing loss of solvent from the reaction mixtures when the samples are being incubated at temperatures near their boiling point A heated platen covers the tops of the sample tubes and is in contact with an individual cap which provides a gas-tight ~eal for each 10 sample tube The heat from the platen heats th- upper parts of each ~ample tube and the cap to a-temperature above the condensation point cuch that no conden-ation and refluxing occurs within any sample tube Condensation represents a relatively large heat transfer since an ~mount of heat equal 15 to the heat of vaporization i- given up when water vapor condenses This could cause large temperature variations from ~ample to sample if the condensation does not occur uniformly The heated platen prevents any condensation from occurring in any sample tube thereby minimizing this source 20 of potential temperature errors The use of the heated platen also reduces reagent consumption Furthermore, the h-ated platen provides a downward force for each cample tube which exceeds an experimentally determined minimum downward force necessary to keep all 25 sample tubes pressed firmly into the temperature controlled sample block ~o a~ to ~stabli~h and maintain uniform block-to-tube tbermal conductance for ~ach tube This uniformity of thermal conductance i~ e~tabli~h-d regardles~ of variations from tube to tube in length, diameter, angle or 30 other dimensional errors which otherwi~e could cause some sample tubes to fit more snugly in their corresponding sample wells than other sample tubes The heated platen ~oftens the pla~tic of each cap but does not totally destroy the caps elasticity Thus, a 3s minimum threshold downward forced i- successfully applied to eac~ tube despite differences in tube height rrom tube to tube The PCR instrument described herein reduce6 cycle times by a factor of 2 or more and lowers reagent cost by accommodating PCR volumes down to 20 uh but remains 5 compatible with the industry 6tandard 0 5 ml microcentrifuge tube Brief Description of the Dr~wincs Figure 1 is a block diagram of the ther~al cycler according to the teaching~ of th- inv-ntion Figure 2 is a plan view of a ~ample block according to the teachings of the invention Figure 3 is a side, elevation view of the sample block showing the bias and ramp cooling channel~
Figures 4 and 5 are ~nd, el-vation view~ of th- sample lS block Figure 6 is a cectional view of the ~ample block taken along cection line 6-6' in Figure 2 Figure 7 i8 a cectional view of the ~ample block taken along section line 7-7' in Figure 2 Figure 8 is a sectional view of the ~ample block taken along cection lin- 8-8' in Figure 2 Figure 9 i- a cros~ ctional, elevation view of the sample block structure after acse~bly with the three-zone film heater and block support Figure 10 i~ a graph of po~er line voltage illuctrating the form of power control to th- three-zone film heater Figure 11 is a temperature graph showing a typical three incubation temperature PCR protocol Figure 12 i~ a cros~-c-ctional view of the sample block 30 illustrating the local zone conc~t Figure 13 is a plan view of the thr~e-zone h-ater Figure 14 is a graph of ~ample temperature vercu6 time illustrating the effect of an r of a sample tube seating force F which i6 too low Figure 15 is a cross-sectional view of a sample tube and cap ceated in the sample block Figure 16A is a grap~ of the impul-e r--pon-e of an RC
circuit Figure 16B is a graph of an impul-e excitation pulse Figure 16C is a graph illustrating how the convolution S of the thermal impulse response and the temperature history of the block give the calculated sample temperature Figure 16D illustrates the electrical analog of the thermal response of the sample block/s~mple tube system Figure 17 illustrates how the calculat-d temperatures 10 of six different camples all converge on a target temperature to within about 0 5'C of- ~ach other when the constants of proportionality for th- ~quations used to control the three zone heater are properly aet Figure 18 i- a graph illu-trating how the denaturation 15 target temp-rature affects th- amount of DNA generated Figure 19 i- a croc----ctional view of th- cliding cover and heated platen Figure 20 is perspective view of the sliding cover, sample block and the knob used to lower the heated platen Figure 21A is a cross-sectional view of the a~embly of one embodiment of th- frame, r-tainer, sample tube and c~p when ~eated on a sample block Figure 21B i- a cro~-sectional vi-w of the assembly of the preferred e~bodiment of the frame, retainer, sample tube 25 and cap when s-ated on the ~ample block Figure 22 $s a top, plan vi-w of the pl~stic, disposable frame for the microtiter plate Figure 23 i- a bottom, plan vi-w of the frame Figure 24 is an end, ~levat$on v$-w of the frame Figure 25 i- another end, ~levation vi-w of the frame Figure 26 i~ a cros--~ectional view of the frame taken along ~ection line 26-26' in Figure 22 Figure 27 is a cro~s-sectional view of the frame taken along ~ection line 27-27' in Figure 22 Figure 28 is an edge elevation view and partial section of the frame Figure 29 is a ~ection~l view of the pr-f-rred ~ample tube Figure 30 is a sectional view of the upper part of the sample tube Figure 31 is an elevation view of a portion of the cap strip Figure 32 is a top view of a portion of the cap strip Figure 33 is a top, plan view of th- plastic, disposable retainer portion of tbe 96 w-11 uicrotiter tray Figure 34 is a ~ide, elevation view with a partial section of the retainer Figure 35 is an end, elevation view of th- retainer Figure 36 is a ~ectional view of the retainer taken along ~ection line 36-36' in Figure 33 Figur- 37 i- a ~ectional view of the retainer taken along ~ection line 37-37' in Figur- 33 Figure 38 is a plan view of the plastic di~posable support base of the 96 well microtiter tray Figure 39 is a bottom plan view of the bas-Figure 40 i5 a side el-vation view of tb- base Figure 41 i~ an ~nd elevation view of tb- ba-e Figure 42 i- a cectional vi-w of the ~upport base taken along ~ection lin- 42-42' in Figur- 38 Figure 43 i- a ~ectional vi-w of the support base taken 25 along ~-ction linc 43-43' in Figure 38 Figure 44 i- a ~ection view of the ba-e t~ken along section line 44-44' in Figure 38 Figur- 45 i~ a p-r~pectiv- exploded view of the plastic dispo~able items that comprise the uicrotiter tray ~ith ~ome 30 ~ample tube- and caps in place Figure 46 i- a diagram of tbe coolant control ~y~tem 24 in Figure ~
Figures 47A and 47B are a block diagram of the control electronics according to the t-achings of th- inv~ntion Figure 48 i8 a 6chematic of a typical zener temperature sensor Figure 49 is a time line diagram of a typical sample period.
Figure 50 is elevation sectional view of a tall thin walled sample tube marketed under the trademark MAXIAMP.
Figure 51 is a graph showing the difference in response time between the thin walled sample tubes and the thick walled prior art tubes.
Figure 52 is a plan view of a sample tube and cap.
Figures 53 and 54 are flow charts of the power up test sequence.

Detailed Description of the Invention Referring to Figure 1 there is ~hown a block diagram of the major system components of one embodiment of a computer directed instrument for performing PCR according to the teachings of the invention. Sample mixtures including the DNA or RNA to be amplified are placed in the temperature-programmed sample block 12 and are covered by heated cover 14.
A user supplies data defining time and temperature parameters of the desired PCR protocol via a terminal 16 including a keyboard and display. The keyboard and display are coupled via bus 18 to a control computer 20 (hereafter sometimes referred to as a central processing unit or CPU). This central processing unit 20 includes memory which storès the control program described below, the data defining the desired PCR protocol and certain calibration constants described below. The control program causes the CPU 20 to control temperature cycling of the sample block 12 and implements a user interface which provides certain displays to the user and which receives data entered by the user via the keyboard of the terminal 16.
In the preferred embodiment, the central processing unit 20 is custom designed. A block diagram of the electronics will be discu-~ed in more detail below In alternative embodiments, the central processing unit 20 and associated peripheral electronics to control the various heaters and other elcctro-mechanical sy~tems of the 5 instrument and read various ~ensors could be any general purpose computer such as a suitably programmed personal computer or microcomputer The samples ~0 are stored in capped disposable tubes which are ~eated in the cample block 12 and are thermally lo isolated from the ambient air by a heat-d cover 14 which contacts a plastic di~posable tray to be de~crib d below to form a heated, enclosed box in which the ~ample tubes reside The heated cover serves, among other things, to reduce undesired h-at transfer- to and from the ~ample 15 mixture by evaporation, condenF~tion and refluxing inside the sample tubes It also r-duces the chance of cross contamination by keeping the insides of the caps dry thereby preventing aerosol formation when the tubes are uncapped The heated cover i- in contact with th- sample tube caps and 20 keeps them h-ated to a temperature of approximately 104~C or above the condensation point~ of the variou~ componcnts of the reaction mixture The c-ntral proces~ing unit 20 includes appropriate electronics to cense the temperatur- of the heated cover 14 25 and control ~l-ctric re-i~tance h~ater- th-rein to maintain the cover 14 at a pr-determin-d temperature Sensing of the temperature of the heat-d cover 14 and control of the resi~tance h-ater- therein i- accompli-h-d via a temperature ~ensor (not ~hown) and bu~ 22 A coolant control ~y~tem 24 continuou~ly circulates a chilled liguid coolant ~uch a- a mixture of automobile antifreeze and water through bias cooling channels (not shown) in the ~ample block 12 via input tubes 26 and output tube 28 The coolant control ~ystem 24 also controls fluid 35 flow through higher volume ramp cooling fluid flow paths (not shown) in the sample block 12 Tbe ramp cooling channels are used to rapidly change the temperature of the sample block 12 by pumping large volumes of chilled liguid coolant through the block at a relatively high flow rate Ramp cooling liquid coolant ~nter~ the ~ampl- block 12 5 through tube 30 and exits the sample block through tube 32 The details of the cool~nt control system are ~hown in Figure 46 The coolant control ~y~tem will be discussed more fully below in the description of th- el~ctronic~ and 60ftware of the control ~ystem Typically, the liquid coolant uc-d to chill the aample block 12 consists mainly of a mixture of water and ethyl-ne glycol The liguid coolant is chilled by a heat exchanger 34 which receives liquid coolant which has extracted heat from the eample block 12 via input tub~ 36 The heat 15 exchanger 34 recei~es comprea-ed liquid freon refrigerant via input tube 38 from a refriger_tion unit 40 This refrigeration unit 40 includes a compressor (not ~hown), a fan 42 and a fin tube heat radiator 44 The refrig-ration unit 40 compresses fr-on gas r-ceived from the heat 20 exchanger 34 via tube 46 The ga~-ous fr-on is cooled and condensed to a liquid in the fin tube condens-r 44 The pr-~sur- of the liquid freon is maintained ablov- its vapor pressure in the fin tube condenser by a flow restrictor capillary tube 47 The output of thi- capillary tube i~
25 coupl-d to the input of the heat exchang-r 34 via tube 38 In the heat exchanger, the pre~sure of th- fr-on i- allowed to drop below the freon vapor pr--~ure, and the fr-on expands In thi- ~o~ ~- of -Yran-ion, h-at ia ab~orbed from the warm-d liquid coolant circulating in the heat 30 exchanger and this heat i- transferr-d to the fr-on thereby causing the freon to boil The warmed freon is then extracted from the heat exchanger via tube 4C and is compres6ed and again circulated through the fin tube condensor 44 The fan 42 blows air through the fin tube 35 condensor 44 to cause heat in the freon from tub- 46 to be exchanged with the ambient air As sy~boliz-d by arrows 48 Th- r-frigeration unit 40 ~hould b~ capabl- of ~xtracting 400 watts of heat at 30~C and 100 wattc of heat at 10~C fro~
the liquid coolant to ~upport the rapid t-mperatur- cycling according to the teachings of the invention S In the preferred embodiment, the apparatus of Figure 1 is enclosed within a housing (not ~hown) The heat 48 expelled to the ambient air is kept within the housing to aid in evaporation of any condensation which occurs on the various tubes carrying chill-d liguid-coolant or ~r-on from 10 one plac- to another This cond-n~tion can cau~- cG~.o~ion of metals used in the conctruction of the un$t or the electronic circuitry and should be removed Expclling the heat 48 inside the enclocure h-lps evaporate any condensation to prevent co~c~ion After exchanging itc heat with the freon, the liquid coolant exits the h-at ~Yr~anger 34 via tube 50 and reenters the coolant control cystem where it i8 gated ac needed to the cample block during rapid cooling portionc of the PCR
cycle defined by data entered by the u-er via terminal 16 As noted above, the PCR protocol involvec incubations at at l-a-t two differ-nt t~mperatures and often three diff-rent te~peratures A typical PCR cycle ic chown in Figure 11 with a d-naturation inc~hation 170 done at a te~perature near 94~C, a hybridization inc~hation 122 done 25 at a temperatur- n-ar room t-mperatur- (25 C-37'C) and an extencion incubation 1~4 don- at a t-mperatur- near 50 c Thece t-mperaturec ar- ~ub~tantially diff-rent, and, therefor- meanc muct be provided to move the t-mperature of the reaction mixtur- of all t~e ~ampl-- rapidly fro~ one 30 temperature to another The ramp cooling ~ystem ic the meanc by which the temperature of the cample block 12 is brought down rapidly from the high t-mperature d-naturation incubation to the lower temperatur- hybridization and extension incubation te~perature- Typically th- coolant 35 temperature ic in the range from 10-20 C When the coolant is at 20 C it can pu~p out about 400 wattc of heat fro~ the 6ample block Typieally the ramp cooling channel dimen-ions, coolant temperatur- and coolant flow rat- are ~et ~uch that peak cooling of S -6 C per ~-cond can be achieved near the high ~nd of tb- operat$ng range (lOO C) 5 and an average cooling rate of 2 5 C per ~econd i- achieved in bringing the eample block temperature down from 94 C to 37~C
The ramp cooling ~y~tem, in ~ome embodim~nt~, may al60 be u~ed to naintain the ~ampl- block t-op-ratur- ~t or n-ar lO the target incubation t~mperature al~o u~ r, in the preferred ~mbodiment, ~mall temparatur~ chang-- of the sampl- block 12 in the downward direction to naintain target incubation temperature are inplement-d by th- bia- cooling system As eeen in Figure 46, a pump 41 con~tantly pumps coolant from a filt~r/r--ervoir 39 (130 milliliter capacity) via 1/2" pipe and pumps it via a 1/2~ pipe to a branching inter~ection 47 Th- pump 41 ~upplie~ coolant to pipe 45 at a constant flow rat~ of 1-1 3 gallon~ per minute At the 20 inter~-ction 47, a portion of th- flow in tube 45 i~
diverted as th- con~tant flow through th- bia~ cooling channels 49 Another portion of the flow in tube 45 is diverted through a flow restrictor 51 to ou~puL tube 38 Flow restrictor Sl ~aintains ~ufficient pr-~sure in the 25 ~y~tem ~uch that a po~itive pre-~ure exi~t~ at the input 53 of a two ~tat- ~olenoid operat-d valve 55 under th- control of the CPU 20 via bu~ S4 Wh-n ra~p cooling i~ d-~ired to implement a rapid downward temperatur~ ng~, th- CPU 20 cause~ the ~olenoid operated valve SS to open to allow flow 30 of coolant through th- ramp cool$ng channal~ S7 There ar-8 ramp cooling channel~ so the flow rate through ~ach ramp cooling channel i~ about 1/8 gallon per ninut- The flow rate through the bia~ cooling channel~ i~ nuch le~a b cau~e of the greatly restrict-d cro-----ctional ar-a th-r-of The bia~ cooling ~ystem provide~ a ~mall con~tant flow of chilled coolant through bia- cooling chann~ 9 in the CA 022660l0 l999-04-Ol sample block 12. This cau-e~ a con-tant, ~~all heat 10~6 from the cample block 12 which i- compen~at-d by a ~ulti-zone heater 156 which i- thermally coupl-d to the ~ample block 12 for incubation ~egments wher- the temperature of 5 the sample block is to maintained at a ~teady value The constant ~mall heat loss caused by the bias cooling flow allows the control ~ystem to implement proportional control both upward and downward in tenp~ratur- for ~mall temperature~ Thi- r-an- both heating ~n~ cooling at 10 controlled, predictable, ~mall rate~ i- availabl- to the temperature servo ey~tem to corr-ct for block temperature errors to cause the block temperature to faithfully track a PCR temperature profile ent-red by t~e u~er ~he alternative would be to cut off power to the film h-ater and 15 allow the ~a~pl~ block to cool by giving up heat to the ambient by radiation and corv~ ion when the block temperature got too high Thi~ would be too ~low and too unpredictable to ~e-t tight t-~perature control epecification- for ~uantitativ- PCR cycling This multi-zone h-ater 156 ic controlled by the CPU 20 via bu- 52 in Figur- 1 and i- the ~eans by which the temperatur- of th- ~ampl~ block 12 i- rai~-d rapidly to higher incubation tenp-ratures from lower incubation temperature~ and i- the ~-an- by wh$ch bia- cooling is 25 comp-n~ated and t-~p ratur- ~rror~ ar- CG~ t-d in the upward dir-ction during t-mp~ratur- tracking and control during incubation~
ln alternativ- ~mbodi~-nt~, biae cooling ~ay be eliminated or ~ay be ~uppli-d by oth-r ~-an~ ~uch ae by the 30 uee of a cooling fan and cooling fin~ forD-d in th- ~etal of the cample block, pelti-r ~unction~ or constantly circulating tap water Car- ~u~t b- t~ken however in these alternative embodi~ent- to in~ur- t~at te~p-ratur- gradients are not created in the ~ample block which would cau6- the 35 temperature of ~ome ~amples to diverg- from the temperature of other 6amples thereby poe-ibly causing different PCR

ampl i f ication re5ults in ~ome ~ampl- tube- than in others In the preferr-d ~mbodim-nt, th- bia5 cooling i~
proportional to the diffcrence between the block temperature and the coolant temperature S The CPU 20 controls the temperature of the ~ample block 12 by sensing the temperature of the metal of the 6ample block via temperature ~ensor 21 and bus 52 in Figure 1 and by ~ensing the temperature of the circulating coolant liquid via bus 54 and a temperatur- ~en-or in th- cool~nt COI-~ ol 10 system The temperature aensor for thc coolant i- abown at 61 in Figure 46. The CPU also ~en~e6 tb- int-rnal ambient air temperature within the houaing of the ~y~tem via an ambient air temperature ~ensor 56 in Figur- 1 Fusther, the CPU 20 ~ense~ the line voltag- for th- input power on line 15 58 via a s-n~or ~ymbolized at 63 All the-e item~ of data together with item- of data ~ntered by th- u~er to define the de~ired PCR protocol ~uch a- targ-t temperature6 and times for incubations are used by a control program to be described in more detail below This control program 20 calculates the amount of power to apply to t~e various zones of the ~ulti-zon- ~ampl- block film h-ater 156 via tbe bus 52 and generat-- a coolant cGr~ol ~ignal to op-n or clo~e the ~olenoid op-rated valv- SS in th- coolant control ~ystem 24 via bus 54 ~o a~ to cau-- th- t mperature of the ~ample 25 block to follow th- PCR protocol d-fln-d by data ent-r-d by the u--r R-ferring to Figur- 2, th-r- i- ahown a top vi-w of the ~ample block 12 The purpo-- of th- aa~pl- block 12 is to provide a ~echanical ~upport and h-at ~ch~~ge ~lem-nt for 30 an array of tbin wall-d ~a~pl- tub-- ~h-r- h-at may be exchanged between th- ~ample liquid in each ~ample tube and liguid coolant flowing in th- bia- cooling and ra~p cooling channels form-d in the aampl- block 12 Fur~b-r, it is tbe function of tbe ~ample block 12 to provide thi~ h-at 35 exchange function without cr-ating large te~perature gradients between various ones of tbe ~ample wells cuch that all sample mixtures in the array experience the eame PCR
cycle even though they are ~patially ~eparated It is an overall objective of the PCR instrument described herein to provide very tight temperature control over the temperature 5 of the sample liquid for a plurality of ~amples such that the temperature of any 6ample liguid does not vary appreciably (approximately plus or minus 0 5 C) from the temperature of any other ~ample liquid in another well at any point in the PCR cycle There is an emerging branch of PCR technology called "quantitative" PCR In this technology, the objective is to perform PCR amplification as precisely as possible by causing the amount of target DNA to ~xactly double on every cycle Exact doubling on every cycle i~ difficult or 15 impossible to achiev- but tight temperature control helps There are many ~ources of error6 which can cause a failure of a PCR cycle to exactly double the amount of target DNA (hereafter DNA ~hould be understood as also referring to RNA) during a cycle For example, in ~ome PCR
20 ~mplifications, th- proce-- ~tart- with a ~ingle cell of target DNA An error that can ~asily occur re~ults when this ~ingle c-ll ~tick~ to the wall of the ~ample tube and does not amplify in the fir~t ~everal cycles Another type of error i~ the entry of a foreign 25 nuclease into the reaction mixture which attacks the ~foreign~ target DNA All cell~ have ~ome nonspecific nuclease that attacks foreign DNA that i5 loose in the cell When thi~ happen~, it int-rfere~ with or atops the replication proce~s Thu-, if a drop of aaliva or a 30 dandruff particl- or material from another ~ample mixture were inadvertently to enter a ~ampl- ~ixtur-, the nuclease materials in these cell~ could attack the target DNA and cause an error in th- amplification process It i~ highly desirable to eliminate all ~uch ~ources of cross-35 contamination Another source of error is nonpreci-e control over sample mixture temperature as betwe-n various ones of a multiplicity of different ~amples For ~xample, if all the samples are not precisely controlled to have the proper annealing temperature (a user ~elected temperature usually 5 in the range from 50 to 60~C) for the extension incubation certain forms of DNA will not extend properly This happens because the primers used in the exten~ion proces6 anneal to the wrong DNA if the temperature iB too low If the annealing temperature i~ too high, the primers will not 10 anneal to the target DNA at all One can easily imagine the consequences of performing the PCR amplification proce~ inaccurately wben PCR
amplification is part of diagno~tic t-~ting ~uch a- for the presence RIV antibodies, h-patiti~, or the pre6ence of 15 genetic diseases ~uch as sickle cell anemia, etc A fal~e positive or false negative r-~ult in ~uch diagno~tic testing can have disastrous personal and legal conse,~ences Accordingly, it i~ an object for the de~ign of the PC~
instrument describ d berein to eliminate as many of these 20 ~ources of po6sible ~rror- a~ po~-ible ~uch as cross-contamination or poor temperatur- control while providing an instrum-nt which i~ compatibl- with th- indu~try ~tandard 96-well microtiter plate fornat The in~trument must rapidly perform PCR in a flexible manncr witb a ~imple user 25 interface ~ n the preferred ~mbodiment, the ~ample block 12 is machin- out of a ~olid block of relatively pur- but corro~ion re~i~tant aluminu~ ~uch ~ the 6061 aluminum alloy Machining the block ~tructure out of a ~olid block 30 of aluminum result~ in a ~ore tbermally homogenous structure Cast aluminum ~tructur~s tend not to be as thermally homogenous as is nec~ss~ry to meet the very tight desired temperature control ~p-cifications Sample block 12 i- capable of rapid change~ in 35 temperature because the thermal mas6 of the block is kept low This is done by the formation in the block of many cooling passageways, sample wells, grooves and other threaded and unthr-aded holes Some of th~se hol~s are u-ed to attach the block to supporte and to attach ~xternal devices euch as manifolds and ~pillage traye ther-to To best appreciate the ~honeycomb" nature of the eample block structure, the reader ~hould refer simultaneously to Figure 2 which shows the block in plAn view ~s well ~s Figures 3 through 8 which ~how elevation views and strategically located ~ctional VieW8 of the ~ampl- block 10 For exAmple, Figure 3 is a ~ide elevation vi-w ~howing the cooling channel positions taken fro~ the vantag- point of the view line 3-3' in Figur- 2 The ~levation view of the sample block 12, looking at the oppo-ite ~dge, i- identical Figure 4 is an ~l-vation view of th- ~dge of the eample ~5 block 12 from the perep-ctive of view line 4-4' in Figure 2 Figure 5 is an ~levation view Or the end of th- ~ample block 12 taken from the p-rspective of vi-w line 5-5' in Figure 2 Figure 6 is a ~ectional view of ~e ~ample bloek 12 taken along the section lin- 6-6' in Figur- 2 Figure 7 is a 20 sectional view of th- ~ampl- block 12 taken along eection line 7-7' in Figur- 2 Figure 8 i- a eectional view of the sampie block 12 taken along e-ction line 8-8' in Figure 2 The top surface of the eample block 12 is drilled with an 8 x 12 array of conical eample wells of which wells 66 25 and 68 are typical Th- conical configuration of each sample well is beet ~n if Figure 8 Th- walls of each sample well are drill-d at an angl- of 17 to match th-angle of the conical e-ction of ~ach ~ampl- tub~ Thl- i-done by drilling a pilot hole having the diam-ter D~ in 30 Figure 8 Then a 17 countereink is ue-d to form the conical walls 67 The bottom of each ~ample well includes a ~ump 70 which has a depth which ~xce-de th- depth of penetration of the tip of t~e cample tube The eump 70 ie creat-d by the pilot 35 hol- and provides a small open ~pac- b n-ath the aample tube when the eample tube is seated in the correeponding ea~ple well This 6ump provides a ~pace for liquid such as condensation that forms on the well walls to reside without interfering with the tight fit of ~ach ~ample tube to the walls of the ~ample well Thi- tight fit i- necess~ry to 5 insure that the thermal conductance from the well wall to the sample liquid is uniform and high for each cample tube Any contamination in a well which causes a loose fit for one tube will destroy this uniformity of thermal conductance across the array That i-, becaus~ liquid i- cub~tantially 10 uncompressible at the pres-ures involv-d in ~-ating the sample tubes in the sample wells, if there were no ~ump 70, the presence of liquid in th- bottom of the ~anplc well could prevent a ~ample tube from fully s-ating in its ~ample well Furthermore, the sump 70 provides a ~pace in which a 15 gaseous phase of any liquid re-iding in the sump 70 can expand during high t-mperature incubations ~uch that large forces of ~uch expansion which would be present if there were no sump 70 are not applied to the ~ample tube to push the tube out of flu~h contact with th- ~ampl- well It has been found experimentally that it i~ important for each sample tube to be in flush contact with its corresponding ~ample well and that a certain minimum threshold force be applied to each ~ample tube to keep the thermal conductivity b twe-n the wall~ of the ~ample well 25 and the reaction ~ixtur- uniform throughout th- array This minimum thr-shold ~eating force i- ~hown as th- force vector F in Figure 15 and i~ a key factor in pr-venting th- thermal conductivity through the wall~ of on- ~ample tub- from being different than the thermal conductivity through the walls of 30 another sample tube located elsewhere in the block The minimum threshold seating force F is 30 grams and the preferred force level ~s b tween 50 and 100 grams The array of ~ample well~ ubstantially eompletely surrounded by a groove 78, best ~een in Figures 2, 6 and 8, 35 which has two functions The main function i- to reduce the thermal conductivity from the central area of the sample block to the edge of the block The groove 78 ~xtends about 2/3 through the thickness of the ~ample block Thi- groove minimizes the effects of unavoidable thermal gradients caused by the neceF6~ry mechanical connection~ to the block 5 of the support pins, manifolds, etc A secondary function is to remove thermal mass from the sample block 12 SO as to allow the temperature of the sample block 12 to be altered more rapidly and to simulate a row of wells in the edge region called the "guard bandn The amount of ~-tal r moved 10 by the portion of the groove 78 betw--n points 80 ~nd 82 in Figure 2 iS designed to be substantiaily equal to the amount of metal removed by the adjacent column of ~ight ~ample wells 83 through 90 The purpose of this is to match the thermal mass of the guard band to th- thermal ~ass of the lS adjacent "local zone~, a term which will be explained more fully below Referring specifically to Figur-s 3, 6 ~nd 8, there i~
~hown the number and relative po-ition~ of the various bias cooling and ramp cooling channels which are forced in the 20 metal of the sample block 12. There are nin- bias cooling channels marked with reference numerals 91 through 99 Likewise, there are cight ramp cooling channel~ marked with reference numeral~ 100 through 107 Each of these bia- cooling and ramp cooling channels is 25 gun drilled through the aluminum of the sample block The gun drilling proces- is well known and provides th- ability to drill a long, very straight hol- ~hich is as clo~e as possible to the bottom surface 110 of the sampl- block 12.
Since the gun drilling process drills a straight hole, this 30 process i8 preferred so as to pr-vent any of the bia-cooling or ramp cooling chann-ls from ~traying during the drilling process and penetrating the bottom surface 110 of the sample block or oth-rwi-- altering its position relative to the other cooling chann-ls Such mispositioning could 3s cause undesirable t~ ~rature gradients by ups-tting the "local balance" and "local symmetry" of the local zones These concepts are explained below, but for now the reader should understand that these notion~ and the ~tructures which implement them are key to achieving rapid temperature cycling of up to 96 camples without creating excessive 5 temperature errors as between different cample wells The bias cooling channels 91 through 99 are lined with silicone rubber in the preferred embodiment to reduce the ther~al conductivity acroc~ the wall of the bias cooling channel Lowering of the ther~al conductivity ~cro~- the 10 channel wall in the bias cooling channel~ ~- pr-f-rred co as to prevent too rapid of a change in t-mperature of the sample block 12 when the multi-zone heater 156 is turned off and heat loss from the ~ample block 12 i primarily through the bia~ cooling channels This is the ~ituation during the lS control process carried out when the ~ample block temperature ha~ etrayed elightly abov- the desired target incubation temperature and the control cyctem is attempting to bring the cample block temperature back down to the user's epecifi-d incubation temperature Too fast a cooling 20 rate in this eituation could cause overshoot of the desired incubation temperature befor- the control cyetem'c ~ervo feedback loop can re-pond although a ~controlled overshoot"
algorithm is u~ed a~ will be described below Sinc- the block temperature ~ervo feedback loop has a time constant 25 for reacting to ctimuli, it ic d-eirable to control the amount of heating and cooling and the r--ulting rate of temperature change of the cample block cuch that overshoot is minimized by not changing the cample block t-mperature at a rate fa~ter than the control cyet-m can respond to 30 temperature error-In the preferred embodim-nt, the bia~ cooling channels are 4 millimeters in diameter, and the eilicone rubber tube has a one millimeter in~ide diameter and a 1 5 millimeter wall thickness ThiS providee a biae cooling rate of 35 approximately 0 2 C per cecond when the block is at the high end of the operating range, i e , n-ar lOO C, and a bias cooling rate of approximately O l C per ~econd when the sample block 12 is at a temperature in the lower end of the operating range The coolant control system 24 in Figure 1 causes a flow rate for coolant in the bias cooling channels s of approximately 1/20th to 1/30th of the flow rate for liguid coolant through the ramp cooling channels, lOo through 107 The bias cooling and ramp cooling channels are the same size, i e , 4 millimeters in diameter, and extend completely through the ~ample block 12.
The bias cooling channels are lined by inserting a stiff wire with a hook at the end ther-of t~rough the bias coolinq channel and hooking it through a hole in the end of a silicone rubber tube which has an outside diameter which is ~lightly greater than 4 millimeter- The hook in the 15 wire i6 then placed through th- hole in the ~ilicone rubber tube, and the silicone tube i- pulled through the bias cooling channel and cut off flush with the end eurfaces of the sample block 12 Threaded holes 108 through 114 are used to bolt a 20 coolant manifold to each eide of the eample block 12 There is a coolant manifold bolted to ~ach end of the block These two coolant ~anifolds are coupled to the coolant channels 26, 28, 30 and 32 in Figure 1, and are affixed to the sample block 12 with a gasket material (not shown) 25 interpo-ed between the manifold and the eample block metal This gaeket prevents leaks of coolant and limite the thermal conductivity between the ~ample block 12 and the manifold which represents a h-at eink Any gaeket material which s-rves the above etated purposes will ~uffice for practicing the invention The positions of the biae cooling and ramp cooling channels relative to the position of the groove 78 are best 35 seen in the sectional view of Figure 6 The positions of the bias cooling and ramp cooling channels relative to the positions of the sample wells is best ~een in Figure 8 The bias cooling and ramp cooling channels are generally interposed between the positions of the tips of the sample wells Further, Figur- 8 reveals that th- bias cooling and 5 ramp cooling channels such as channels 106 and 97 cannot be moved in the positive z direction very far without risking penetration of the walls of one or ~ore ~ample wells Likewise, the cooling channels cannot be ~oved in the negative z direction very far wlthout croating the 10 possibility of penetrating the bottom ~urfac- 116 of the sample block 12 For clarity, the position- of the biac and ramp cooling channels are not ~hown in hidden lines in Figure 2 relativ- to th- positions of the ~ample wells and other ~tructures However, there i- either a bias cooling 15 channel or a ramp cooling channel b tween every column of sample wells Referring to Figure 2, the holes 118, 119, 120 and 121 are threaded and are used to attach the cample block 12 to machincry u~ed to ~achin- the various hol-s and grooves 20 formed therein In Figur-s 2, 4 and 5, the hole- 124, 125, 126 and 127 ar- used to attach the ~ample block 12 to a support bracket ~hown in Figur- 9 to b~ describ~d in more detail below St-el bolts ext-nd through this support bracket into the thr-aded holes 124 through 127 to provide 25 mechanical ~uppGL~ of the ~ample block 12 Th-se steel bolts also represent he~t sink- or heat sourc-- which tend to add thermal ~a-s to the ~ampl- block 12 and provide additional pathways for transfer of thermal energy between the sample block 12 and the ~u~,o~lr~ng onvironment These 30 support pins and the ~anifolds ar- two i~portant factors in creating the need for the guard bands to prevent the thermal energy transferred back and forth to these peripheral structures from ~ffecting these ~ampl- t-mp-ratures Referring to Figure 5, the holes 128, 130 and 132 are 35 mounting holes for ~n integrated circuit temper~ture sensor (not shown) which is inserted into the sample block through CA 022660l0 l999-04-Ol hole 128 and secured thereto by bolts which fasten to threaded holes 130 and 132 The extent of penetration of the hole 128 and the relative po~ition of the temperature sensor to the groove 78 and the adjacent column of ~ample 5 wells is best seen in Figure 2 Referring to Figure 2, holes 134 through 143 are mounting holes which are used to mount a spill collar 147 (not shown) This spill collar 147 is shown in Figure l9 detailing the structure of the heated platen 14, sliding 10 cover 316 and lead screw assembly 312. The pu~Gse of the spill collar is to prevent any li~uid ~pill-d from the sampl- tubes from getting in~ide the instrum-nt casing where it could cause corrosion Referring to Figure 9, th-r- i~ shown in cross-~ection 15 a view of the support ~ystem and multi-zone h-ater 156 configuration '~r th- ~ample block 12 . The ~ample block 12 is supported by 'our bolts of which bolt 146 is typical ~hese four bo~s pa~- through upright mcmber~ of a steel support bracket 148 Two large coil aprings ,0 and 152 are 20 compressed between a horizontal portion of the ~upport bracket 148 and a ~t-el pres~ure plate 154 The ~prings 150 and 152 are compressed ~ufficiently to supply approximately 300 lbs per ~quare inch of force in the po~itive z direction acting to compress a film heater 156 to the bottom 25 surface 116 of the ~ample block 12 This three layer film heater ~tructur- i~ comprised of a multi-zone film heater 156, a ~ilicone rubber pad 158 and a laycr of ~poxy resin foam 160 In th- pref-rr-d embodim-nt the fil~ h-ater 156 has three ~eparately controllable zones Th~ purpose of the 30 film heater 156 i- to supply heat to the ~ampl- block 12 under the control of the CPU 20 in Figure 1 The purpose of the silicone rubber pad 158 is to lower the thermal conductivity from the film heater layer 156 to the structures below These lower ~tructure~ ~-rve as heat 35 sinks and heat source~ betw-en which undesired h-at energy may be transferred to and from the aample block 12 The silicone rubber pad 158 has the additional function of compensating for ~urface irr-gularities in the film heater 156 6ince ~ome film heaters ~mbody nichrome wir-s and may not be not perfectly flat The purpo e of the steel plate 154 and the epoxy resin foam 160 is to transfer the force from the ~prings 150 and 152 to the silicone rubber pad 158 and the multi-zone film heater 156 60 as to compress the film heater to the bottom surface 116 of the sample block with as flu-h a fit as 10 possibl- Th- epoxy resin foam ~hould be ~tiff ~o a- to not be crushed under the force of th- ~pring6 but it ~hould al60 be a good insulator and 6hould have low thermal mass, i e , it should be a nondense ~tructure In one embodiment, the foam 160 ic manufactured under the trademark ECK0 foam In alternative mbodiments, other structures may be ~ubstitut-d for the ~ilicone rubber layer 158 and/or the epoxy resin fo~m layer 160 For example, a stiff boneycomb ~tructure ~uch a~ i~ u-ed in airplane 20 construction could be placed between the pres~ure plate 154 and the film h-ater 156 with in6ulating lay-rc therebetween Whatever ctructure is used for layer- 158 and 160 ~hould not absorb 6ubstantial amountc of h-at from the cample block 12 while the block ic being heated and chould not transfer 25 sub~tantial amount~ of b-at to the ~ampl- block 12 when the block i- b~ing cooled Perfect i~olation of the block from it6 ~urrounding 6tructure~ however, i~ virtually impossible Every ~ffort ~bould be made in de~igning alternative structures that will be in contact with the ~ample block 12 30 so as to thermally i~olate the ~ample block from its environment as much a~ possible to minimize the thermal mass of the block and enable rapid temperature changes of the sample block and tbe ~ample mixture~ ~tor-d therein Preci6e temperature control of the ~ample block 35 temperature is achi-v-d by the CPU 20 in Figure 1 by controlling the amount of heat applied to the ~ample block by the multi-zone film heater 156 in Figure 9. The film heater is driven using a modified form of pulse width modulation. First, the 120 volt waveform from the power line is rectified to preserve only half cycles of the same 5 polarity. Then portions of each half cycle ar- gated to the appropriate zones of the foil heater, with the percentage of each half cycle which is applied to the various zones of the foil heater being controlled by the CPU 20.
Figure 10 illustrates onc embodiment of a power control 10 concept for the film heater 156. Figure 10 is a diagram of the voltage waveform of the ~upply line voltage.
Rectification to eliminate the negative half cycle 162 occurs. Only positive half cycles remain of whic~ half cycle 164 iS typical. The CPU 20 and its associated 15 peripheral electronic circuitry then controls the portion of each half cycle which is applied to the various zones of the film heater 156 by selecting a portion of each half cycle to apply according to a power lev-l computed for each zone based upon equations given below for ~ach zone. That is, 20 the dividing line 166 is moved forward or backward along the time axis to control the amount of power to the film heater based upon a n~mber of factors which are related in a special eguation for each zone. The cross-hatched area under the positive half cycle 164 represents the amount of 25 power appli-d to the film heater 156 for the illustrated position of the dividing line 166. A~ the dividing line 166 is moved to the right, more power i- applied to the film heater, and the ~ample block 12 gets hotter. As the dividing line i~ moved to the left along the time axis, the 30 cross-hatched area becomes ~maller and le-s power is applied to the film heater. How the CPU 20 and its associated software and peripheral circuitry control the temperature of block 12 will be described in more detail b low.
The amount of power cupplied to the film heater is 35 continuously variable from 0 to 600 watts. ~n alternative embodiments, the amount of power supplied to t~e film heater 156 can be controlled usinq other schemes such as computer control over the current flow through or voltage applied to a DC film heater or by the zero crossing switching scheme described below In other embodiments, heating control of the sample block 12 may be performed by control over the flow rate and/or temperature of hot gases or hot liquid which is gated through heating control channels which are formed through the metal of the cample block 12 Of course in such 10 alternative embodiments, the number of sample ~ells in the block would have to be reduced ~ince-th-re is no room for additional heating channels in the sample block 12 shown in Figures 2 through 8 Such alternative embodiments could still be compatible with the 96-well microtiter plate format 15 if, for example, every other well wcr- removed to ~ake room for ~ heating channel in the sample block This would provide compatibility only as to the dimensions of such microtiter plates and not as to the simultaneous processing of 96 different samples Care must be taken to preserve 20 local balance and local symmetry in these alternative embodiments In the embodiment described herein, the maximum power that can be dclivered to thc block via the film heater is 1100 watts This limitation arise6 from the thermal 25 conductivity of the block/h-ater int-rface It has been found experimentally that the ~upply of more than approximately 1100 watts to the f$1m heater 156 will freguently cause self-d-struction of th- device Typical power for heating or eooling when controlling 30 block temperatures at or near target incubation temperatures is in the range of plu- or minu- 50 watts Referring to Figure 11, t~ere is shown a time versus temperature plot of a typical PCR protocol ~arge downward changes in block temperature are accomplished by gating 35 chilled liquid coolant through the ramp cooling channels while monitoring the sample block temperature by the temperature sensor 21 in Figure 1 Typically these rapid downward temperature changes are carried out during the ramp following the denaturation incubation 170 to the temperature of hybridization incubation 172. Typically, the user must 5 specify the protocol by defining the temperatures and times in one fashion or another so as to describe to the CPU 20 the positions on the temperature/time plane of the checkpoint~ sy~bolized by the circled inter~-ction~ between the ramp legs and the incubation l-gs Cen-rally, the 10 incubation legs are marked with r-f-r-nc- numeral- 170, 172 and 174 and the ramp~ are marked with reference numerals 176, 178 and 180 Generally the incubation interval~ are conducted at a ~ingle temperature, but in alternative embodiments, they may be ~te~ or continuou~ly ramped to 15 different temperature~ within a range of temp~rature- which is acceptable for performing the particular portion,of the PCR cycle involved That i~, the denaturation incubation 170 need not be carried out at one temperatur- a~ ~hown in Figure 11, but may be carri-d out at any of a plurality of 20 different temperatures within the range Or temperatures acceptable for denaturation In ~ome em~odiments, the us-r may ~pecify th- length ot the ramp ~-gment- 176, 178 and 18 0 . In other e~bodiment-, the u~er may only ~pecify the temperature or temperatures and duration of aach incubation 25 interval, and the in~trument will then move th~ tamp-rature of the ~ample block as rapidly as po-sible b tween incubation temperatures upon the completion of one incubation and thc ~tart of another In the preferred embodiment, the u~er can ~l-o have t~mperature~ and/or 30 incubation times which are difrer-nt for aach cycl- or which automatically increment on every cycle The average power of ramp cooling during a transition from a 95 C denaturation incubation to a 35 C hybridization incubation is more than one kilowatt typically This 35 results in a temperature change for the ~ample block of approximately 4-6 C per second when the block temperature is at the high end of the operating range, and approximately 2~C per second when the block temperature is at the low end of the operating range Generally it is desir_ble to have as high a cooling rate as possible for ramp cooling Because so much heat is being removed from the sample block during ramp cooling, temperature gradients across the sample block from one end of a ramp cooling channel to the other could occur To prevent this and minimize these types of temperature gradients, the ramp cooling channels are 10 directionally interlaced That is, in Figure 3, the direction of coolant flow through ramp cooling channels 100, 102, 104, and 106 is into the page as ~y~bolized by the x'~
inside these ramp cooling channel holes Ramp eooling liquid flow in interlaced ramp cooling channels 101, 103, 15 105, and 107 i6 out of the page a- ~ymbolized by the single points in the center of the-e ramp cooling channel holes This interlacing plus the high flow rate through the ramp cooling channels minimizes any temperature gradients which might otherwise occur using noninterlaced flow patterns or 20 lower flow rates because the distances between the hot and cold ends of the channels is made smaller A slower flow rate results in mo~t or all of the heat bQing taken from the block in the first inch or ~o of travel which means that the input side of the block will be at a lower temperature than 25 the output ~ide of the bloc~ A high flow rate ninimizes the t-mperature gradient along the channel Interlacing means the hot end of the rhAnnels running in one direction are ~sandwich-d~ between the cold ends of channels wherein flow is in the opposite direction This is a smaller 30 distance than the length of the chann-l Thus, temperature gradients are reduced because the di-tanres heat must travel to eliminate the temperature gradient are reduced This causes any tcmperature gr_di-nts that form because of cooling in the ramp channels to be quickly eliminated before 35 they have time to differentially heat some samples and not others Without int-rlacing, one side of the sample block would be approximately l C hotter than the other ~ide Interlacing results in dissipation of any temperature gradients that result in less than approximately 15 seconds In order to accurately estimate the amount heat added 5 to or removed from the block, the CPU 20 measures the block temperature using temperature ~ensor 21 in Figure 1 and measures the coolant temperature by way of temperature sensor 61 in Figure 46 coupled to bus 54 in Figure 1 The ambient air temperature i~ also mea6ured by way of lo temperature sen60r 56 in Figur- 1, and th- power line voltage, which controls the power applied to the film heaters on bus 52, i5 al~o m-asured The thermal conductance from the ~ample block to ambient and from the sample block to the coolant are kno~n to the CPU 20 as a 15 result of measurement~ made during an initialization process to set control parameter6 of the ~y~tem For good temperature uniformity of the sample population, the block, at constant temperature, can have no net heat flow in or out However, t-mperature gradients can 20 occur within the sample block ari~ing from local flows of heat from hot 6pot~ to cold ~pot- which have zero net heat transfer relative to the block border~ For instance, a slab of matcrial which i~ heat-d at one end and cooled at the other is at a constant average temperature if the net 25 heat flow into t~e block is z-ro However, in this situation a ~ignificant temperature nonuniformity, i e , a temperature gradient, can be establi~hed within the slab due to the flow of heat from the hot ~dge to the cold edge When heating and cooling of the ~dge~ of th- block are 30 ~topped, the flow of heat from the hot edge to the cold edge eventually dis~ipat-6 thi~ temperature gradient and the block reaches a uniform temperature throughout which is the average between the hot temperature and cool temperature at the beginninq of heat flow If a slab of cross ~ectional area A in length L has a uniform thermal conductivity X, and the ~lab is held at constant average temperature because heat influx from a heat source Qj~ is matched by heat outflow to a heat sink Q~t the steady state temperature profile which results from the heat flow is Qin L
(1) Delta T -A K
Where, Delta T - the temperature gradient L - the thermal path length A ~ the area of the thermal path X - the thermal conductance through the path ~ n general, within any material of uniform thermal conductance, the temperature gradient will be established in 15 proportion to the heat flow per unit area N-at flow and temperature nonuniformity are thus intimately linked Practically speaking, it is not possible to control the temperature of a sample block without ~ome heat flow in and out The cold bias control cooling requir-s ~ome heat flow 20 in from the strip heaters to balance the heat remo~ed by the coolant flowing through the bias cooling channels to maintain the block temperature at a stable value The key to a uniform sample block temperature under these conditions is a geometry which has "local balance" and "local ~y~metry"
25 of heat sources and heat sinks both ctatically and dynamically, and which is arranged such that any heat flow from hot spots to cold apots occur~ only over a hort distance Stated briefly, the concept of ~static local balance"
30 means that in a block at constant temperature where the total heat input cquals the total heat output, the heat sources and heat sinks are arranged such that within a distinct local region, all heat ~ources are completely balanced by heat sinks $n terms of heat flows in and heat 35 flows out of the block Therefore, each local region, if isolated, would be maintained at a constant temperature The conc-pt of "static local symmetry" means that, within a local region and for a constant temperature, the center of mass of heat sources is coincident with the center of mass of heat sinks If thi- wer- not the case, within S each local region, a temperature gradient across each local region can exist which can add to a temperature gradient in an adjacent local region thereby causing a gradient across the sample block which is twice as large as the size of a single local region because of lack of local cymmetry even 10 though local balance within each local r-gion exists The concepts of local balance and local symmetry ar- important to the achievement of a static t-mp-ratur- balance where the temperature of the sample block i8 being maintained at a constant level during, for example, an incubation interval For the dynamic case where rapid temperature changes in the sample block are occurring, the thermal mass, or heat capacity of each local region b comes important ~his is because the amount of heat that must flow into each local region to change its temperature is proportional to the 20 thermal mass of that r-gion Therefore, the concept of static local balance can be expanded to the dynamic case by reguiring that if a local region includes x percent of the total dynamic heat source and heat sink, it must also include x percent of the thermal 25 mass for ~dynamic local balance" to exist Likewise, "dynamic local symmetry" requires that th- center of mass of heat capacity be coincident with the center of mass of dynamic heat aource- and cinks What this means in simple terms is that the thermal mass of the sample block i5 the 30 metal thereof, and the machining of the sample block must be symmetrical and balanced such that the total mass of metal within each local zone is the same Further, the center of mass of the metal in each local zone should be coincident with the center of mass of the dynamic heat sources and 35 sinks Thus, the center of mass of the multi-zone heater 156, i e , its geometric center, and the geometric center of the bias and ramp cooling channels must coincide From a study of Figures 2-9, it will be ~-en from the d-tailed discussion below that both static and dynamic local balance and local sy~metry exi6t in sample block 12 Figure 12 illustrates two local regions side by side for the design of the ~ample block 12 according to the teachings of the invention In Figure 12, the boundaries of two local regions, 200 and 202, are marked by dashed lines 204, 206 and 208 Figure 12 showJ that ~ach local r-gion lo which is not in the guard band is compri-ed of two columns of sample wells; a portion of the foil ~leater 156 which turns out to be 1/8th of the total ar-a of th- heater; one ramp cooling channel ~uch as ramp cooling channels 210 and 212; and, one bias cooling channel To prcserve local 15 symmetry, each local region is centered on its ramp cooling channel and bas one-half on a bias cooling channel at each boundary Fo example, local region 200 has a center over the ramp cooling channel 210 and bia- cooling channels 214 and 216 are dissected by th- local r-gion boundaries 204 and 20 206, r-spectively Thus the center of mass of the ramp cooling channel (the middle thereof)~ coincides (horizontally) with the center of mass of the bias cooling channels (the center of the local region) and with the center of ~ass of the film heater portion coupled to each 25 local region Static local balance will exi-t in each local region vhen the CPU 20 is driving th- film heater 156 to input an amount of h-at ~n-rgy that i- ~qual to the ~mount of heat energy that i- b ing removed by the ramp cooling and bias cooling channelc Dynamic loeal balance for ~ach local 30 region cxists because each local region in the center portion of the block where the 96 ~ample ~ixture~ reside contains approximately 1/8th the total thermal ~ass of the entire sample block, contains 1/8th of the total number of ramp cooling channels and contains l/8th of the total number 35 of bias cooling channels Dynamic local symmetry ~xists for each local reqion, because the center of mass of the metal of each local region is horizontally coincident with the center of film heater portion underlying the local region;
the center of the ramp cooling channel; and, the center of mass of the two half bias cooling channels s By virtue of these physical properties characterized as static and dynamic local balance and local ~ymmetry, the sample block heats and cools all samples in the population much more unifor~ly than prior art thermal cyclers Referring to Figure 2, the plan view of the boundaries lo of the local regions are illu-trat-d by dached lines 217 through 225 Inspection of Figure 2 reveals that the central region of the 96 camplc wells ar- divided into six adjacent local regions bounded by boundaries 218 through 224 In addition, two guard band local regions are added 15 at each edge The ~dge local region (local regions are sometimes herein also called local zones) having the most negative x -oordinate is bounded by boundary lines 217 and 218 The ~dge local region having the most positive x coordinate is bounded by boundary lin-c 224 and 225 Note 20 that the edge local regions contain no ~ample well columns but do contain the groo~e 78 simulating a colu~n of wells The depth and width of the groove 78 is d-signed to remove the same metal mass as a column of welle thereby somewhat preserving dynamic local cymmetry The edge local zones are 25 therefore diff-r-nt in thermal ~as- (they al-o have additional thermal ~ass by virtue of th- external conn-ctions cuch a~ manifolds ~nd support pins) than the six local zones in tbe central part of th- ~ample block This difference is accounted for by heating the edge local zones 30 or guard bands with ~eparately controllable zones of said multizone heater so that more ~nergy may be put into the guard band than the central zone of the block The local regions ~t ~ach edge of the block approximate, but do not exactly match the thermal,properties 35 of the six centrally located local regions The edge local regions are called ~guard band" regions because they CA 022660l0 l999-04-Ol complete a guard band which runs around the periphery of the sample block 12 The purpose of thi- guard band i6 to provide some ther~al isolation of the central portion of the sample block containing the 96 sample wells from S uncontrolled heat ~inks and sources inherently embodied in ~echanical connections to the block by such things as support pins, manifolds, drip collars and other devices which must be mechanically affixed to the sample block 12 For ~xample in Figure 2, the edge surfaces 228 and 230 of 10 the sample block have plastic manifoldc ~ttach-d thereto which carry coolant to and from the ramp and bia- cooling passages The guard band along edges 228 and 230 consists of portions of the slot 78 which are parallel to and closest to the edges 228 and 230 The depth of the groo~e 78 is 15 such that the bottom of the groove i- as close to the perimeters of the bias and ramp cooling channels as is possible without actually int-rs-cting them The width of the groove 78 coupl-d with this depth is such that the volume of metal removed by the slot 78 betwe-n points 82 and 20 232 in Figure 2 approximately equals the volume of metal removed by the adj~cent row of sample wells starting with sample well 234 and ending with samplc well 83 Also, the slot 78 all around the perimeter of the block is located approximately where such an additional row of wells would be 25 if the periodic pattern of ~a~ple well- w-r- ext-nded by one row or colu~n of wells in each direction Along the edges 250 and 252 where the support connections are made to the sample block, the guard band local regions contain, in addition to a portion of the slot 30 78, the full length of several cooling channels Referring to Figure 3, these include 1/2 of a bias cooling channel (e g , 92) which merges with the adjacent 1/2 bias cooling channel of the adjacent local r-gion to form a whole bias cooling channel; a ramp cooling channel (e g , 100); and a 35 whole bias cooling channel (- g , 91) For th- edge local region at edge 250, these cooling channels are 10~, 198 and . . .

99 .
The whole bia- cooling channel~ in the guard bands are slightly displaced inward from the ~dge of the block The reason that these whole bias cooling channel~ are used is 5 because a "half" cooling channel is impractical to build Since the bias cooling channels require 6uch a thick walled rubber lining, it would be difficult to keep a hole through ~ lining of a "half" bia6 cooling channel reliably open Thi6 asym~etry in the ~dge local r-gion~ cau--~ a amall 10 excess los6 of heat to the coolant from the ~dge guard band local regions, but it is ~ufficiently r-mot- from the central region of the ~ample block containing the ~ample wells that its contribution to sample temperature nonuniformities is ~mall Also, since the temperature 15 affects of this ~mall asy~metry are predictablc, the effect can be further minimized by the uce of a ~eparately controllable zone of the multi-zone h-ater ~yctem under each guard band Referring to Figure 13, there are shown three 20 separately controlled zones within the film heater layer 156 in Figure 9 The6~ ~-parately controlled zone~ include edge heater zones which are ~ituated under the guard bands at the exposed edges of the ~ample block 12 which are coupled to the eupport bracket 148 There are also ~eparately 25 controlled ~anifold heater zone~ ~ituated under the guard band~ for the ~dge~ 228 and 230 which ar- attached to the coolant ~anifold- Finally, there i~ a central heater zone that underlies the ~ample wells The power applied to each of these zones i- ~eparately controlled by the CPU 20 and 30 the control ~oftware ~ he film heater 156 i- compo-ed of a pattern of electrical conductor6 formed by ~tchi~g a thin ~heet of metal alloy ~uch a~ ~ncon-l~ The uetal alloy ~elected should have high electrical resi~tance and good re~i~tance 35 to heat The pattern of conductors ~o etched i~ bonded between thin 6heets of an electrically insulating polymeric material such AS Kapton~ Whatever ~aterial i- used to insulate the electrical re~i~tance h-ating el-ment, the material must be resistant to high t~mperatures, have a high dielectric ~trength and good mechanical stability The central zone 254 of the film heater has approximately the same di~ensions as the central portion of the sample block inside the guard bands Central region 254 delivers a uniform power density to the ~ampl- well area Edge heater region- 256 and 258 ar- about a- wide as 10 the edgc guard bands but are not guit- a~ long Manifold heater regions 260 and 262 underlie the guard bands for edges 228 and 230 in Figure 2 The manifold heater zones 260 and 262 are electrically connected together to form one ~eparately controllable 15 heater zone Also, the ~dge h-ater ~ectionc 256 and 25B are electrically coupled together to form a ~-conA ceparately controllable heater zone The third aeparately controllable heater zone i~ the central ~ection 254 Each of thése three separately controllabl- heater zones ha- ~-parate ~l-ctrical 20 leads, and each zone is controlled by a ~eparate control algorithm which may be run on ~eparate microprocessors or a shared CPU as i~ done in the preferred ~mbodiment The edge heater zon-~ 256 and 258 are driven to compensate for heat lost to the ~upport bracket- This heat 25 loss i5 proportional to the temperature dlfference between the ~ample block 12 and the ambient air ~u.~ounding it The edge heater zones 256 and 258 al-o compen~ate for the excess loss of heat from the ~ample block to th- full bia~ cooling channel~ at ~ach ~dge of the block Thi~ heat los- i-30 proportional to the temp-rature diff-r-nce b tween the sample block 12 and the coolant flowing through these bias cooling channels The manifold heater cections 260 and 262 are al~o driven so as to compen-ate for heat lo~t to the plastic 35 coolant manifolds 266 and 268 in Figure 13 which are attached to the edges of the sample block 12 The power for the manifold heater sections 260 and 262 compensates for heat loss which i8 proportional mainly to the temp-rature difference between the ~ample block and the coolant, and to a lesser degree, between the sample block and the ambient 5 air For practical reasons, it i5 not possible to match the thermal mass of the guard band local regions with the thermal masses of the local regions which include the sample wells overlying central heater ~ection 254. For example, lo the plastic coolant manifolds 266 and- 268 not only conduct heat away from the guard band, but they al~o add a certain amount of thermal ~ass to the guard band local regions to which they ~re attached The re~ult of thi~ i~ that during rapid block temperature chang-s, the rat-- of rise and fall 15 of guard band temp~rature do not ~xactly match that of the sample well local regions Thi~ gen-rate~ a dyn~mic temperature gradient between the guard band- and ~ample wells, which if allowed to become large, could per~ist for a time which is longer than i- tol-rable Thi- temperature 20 gradient effect i- roughly proportional to the rate of change of block temperature and i~ minimiz-d by adding or deleting h-at from each guard band local zone at a rate which is proportional to the rate of change of block temper~ture The coefficients of proportionality for the guard band zone he~ter~ are relatively ~t~bl- propertie- of the design of the ~y~tem, and are d-termined by engineering measurement~ on prototype~ The value~ for these coefficients of proportionality ar- giv-n below in 30 connection with the definitions of the terms of Equations (3) through (5) These ~guation~ define the amounts of power to be applied to the manifold heat~r zone, the edge heater zone and the centr~l zone, respectiv-ly in an alternative embodiment The equations used in the preferred 35 embodiment are given below in the description of t~e software (Equations (46)-(48), power distributed by area) (3) P~ - A~ P + X~ (T"~ - T~) + ~2 (TU~ ~ TO~L) + ~3(dt~L~/dt) where, P~ ~ power eupplied to the ~anifold heater zones 260 and 262 A~ ~ ~rea of the manifold heater zone P ~ power needed to cau6e the block temperature to stay at or move to the deeired temperature at any particular time in a PCR thermal cycle protocol - an experimentally d-t-r~ined conctant of proportionality to compeneate for ~YC~e heat loes to a~bient through the ~anifold~, ~gual to 0 watte/ degree Kelvin 15 ~2 ~ an ~xperimentally det~r~ined conetant of proportionality to compensate for excess heat loss to t~e coolant, equal to 0 4 watts/degree Kelvin ~3 - an experimentally det-r~ined con-tant of proportionality to provide extra power to compen~ate for additional tbermal ~ase of the ~anifold ~dge guard band~ caue-d by the attachment of the plaetic ~anifolde etc , egual to 66 6 watt secon~c/degr-e Kelvin 25 T~L~ ' the t-mperature of the ~a~ple block 12 T~4 - the temperature of the ambient air T~L ~ the temperatur- of the coolant dt~/dt - th- change in aample block temperature per unit time (4) PE ~ AEP + KE1 (T~L~ ~ T~) I KE2 (T~L~ ~ To~L) + XE3 (dt,L~/dt) where, PE ~ power to be appli-d to the odge heater zones AE ~ the area of the ~dge heater zones 35 ~E1 ' an experimentally det-rmined con~tant of proportionality to compensate for excess heat 105s to ambient through the manifolds, ~gual to 0 5 watts/degr-e Xelvin R~2 ~ an experimentally determined constant of proportionality to compensate for cxcess heat loss to the coolant, egual to 0.15 watts/degree Kelvin K~3 ~ an experimentally detcrmined constant of proportionality to provide ~xtra power to lo compensate for additional th-rmal ~a-s of the exposed edge guard ~and- caus-d by the attachment of the aacple block 12 to the support pins and bracket, the tcmperature sensor etc , ~qual to 15 4 watt-sec/degree Xelvin (5) Pc ~ Ac P
where Pc - the power to be appli-d to th- central zone 254 of the multi-zon- heater 20 Ac - the area of the central zone 254 .

In each of Eguations (~) through (5), the power term, P is a variable which is calculatcd by the portion of the control algorithm run by the CPU 20 in Figur- 1 which r-ads the u-er defined ~-tpoints and d-teroines what to do next to 2 5 cause the sample block temperatur- to ~tay ~t or b come the proper tcmperatur- to implement th- PCR tcmpcrature protocol defined by the time and t-mperature ~etpoints ~tored in memory by the us-r The manner in which the setpoints are read and the power density i- calculated will be described 30 in more detail below The control algorithm run by CPU 20 of Figure 1 senses the temperature of the cample block via tempcrature sensor 21 in Figure 1 and Figure g and bus S2 in Figure 1 This temperature is differentiated to derive the rate of c~ange of temperature of the ~ample block 12 The CPU then measure~ the temperature of the ambi-nt air via t-~perature sensor 56 in Figure 1 and m-a~ure- the t-mperature of the coolant via the temperatur~ ~-n-or 61 in th- coolant control 5 system 24 ~hown in Figur- 46 The CPU 20 then computes the power factor corresponding to the particular ~egment of the PCR protocol being implemented and makes three calculations in accordance with Equations (3), (4) and (5) by plugging in all the measured t-mperatures, the con-tants of 10 proportionality (which ar- ctQred in nonvolatil- ~mory), the power factor P for that particular iteration of the control program and th- ar-as of th- variou- h-ater zones (which are ctor-d in nonvolatil- u-mory) The power factor is the total power needed to mo~- the block t-mperature from 15 its curr-nt l-v-l to the temperature l-v-l cp-cifi-d by th-user via a cetpoint More details on the calculations performed by the CPU to control heating and cooling are given below in the description of the control coftware HPID
task"
20After the required power to be applied to ~ach of the three zones of the h-ater 156 is calculated, another calculation i- mad- r-garding th- proportion of each half cycle of input power which ic to be applied to ~ach zone in some ~mhodimentc In the preferred ~mbodiment described 25 below, the calculation mode is how ~any half cycles of the total numbQr of half cyclec which occur during a 200 milli-econd campl~ period ar- to b- appli-d to ~ach zone Thi- proce~c i- deccrib d b low in conn~_tion with the discu-sion of Figur-- 47A and 47~ (h-reafter r-f-rr-d to as 30 Figure 47) and the ~PID Task~ of the control coftware In the alternative embodiment cymboliz-d by Figure 10, the computer calculates for ~ach zone, the position of the dividing line 166 in Figur- 10 After thic calculation is performed, appropriate control cignale are generated to 35 cause the power ~uppli-s for th- multi-zone heat-r 156 to do the appropriate switching to cause th- calcul~ted amount of power for each zone to be applied thereto In alternative e ~o~iment~, the multi-zone heater can be implemented using a ~ingle film heater which d-liver~
uniform power density to the entir~ ~ample block, plus one S or two additional film heater~ with only on- zone apiece for the guard bands These additional heaters are ~uperimposed over the single film heater that covers the entire sample block In 6uch an embodiment, only the power necessary to make up the guard band 106se- i6 delivered to the additional lo ~eater zones The power factor P in Equation- (3) through t5) is calculated by the CPU 20 for various point- on the PCR
temperature protocol ba~ed upon the ~-t points and ramp times cpecified by the user How-v-r, a limitation is 15 imposed based upon the maximum power deliv-ry capability of the zone heater mentioned above The constants of proportionality in Eguations (3) through (5) must be properly ~et to adeguately compensate for excess heat lo~e~ in the guard band for good 20 temperature uniformity Referring to Figure 17, there i- ~hown a graph of the difference~ betw-en c~lculated ~ample temperatures for a plurality of different sampl- in respon6e to a ~tep change in block temperature to rai-e the temperature of the sample 25 block toward a denaturation incu~-tion target temperature of approximately 9~ C from a ~ub-tantially lower t~mperature Figure 17 illu~trate- the calculated ~ample liquid temperature~ when th- ~ulti-zone h-at-r lS6 i~ properly managed u-ing the con~tant~ of proportionality given above 30 in the de~initions of the term~ for Equation6 t3) through (5) The various well~ which were u~-d to derive the graph of Figure 17 are indicated ther-on by a single letter and number combination The 8 x 12 well array ~howing Figure 2 is coded by lettered column~ and nu~bered row~ Thu6, for 3s example, ~ample well 90 is al-o designated ~ample well A12, w~ile ~mplc well 89 is also dcsignat-d ~ample well ~12 Lik-wi~e, s~mple well 68 is al-o designat-d ~ample well D6, and 80 on Note that the vell temperature~ aettle in asymptotically at temperatures which are within approximately 0 5 C of ~ach other becauc- of the overall S thermal design described herein to eliminate temperature gradients The foregoing description illustrates how the ~ample block temperature may be controlled to be unifor~ and to be quickly changeabl- Howev-r, in the PCR p~c~ , $t ic the 10 temperature of the ~ample reaction mixtur- and not th- block temperature that is to be y~y~a2med ~n th- preferred embodiment according to the t-aching- of th- invention, the user ~pecifies a ~quence of target temperature~ for ~h~
~mple liouid itself and ~pecifie6 th- $~c~hAtion times for 15 ~he sa~le li~uid at ~ach of ~cse IAr~-t t~Derat~r-s for each ~tag- in th- PCR proces~ The CPU 20 th-n ~anag-~ the ~ampl~ block temperature ~o as to get t~e ~ample reaction mixtures to the ~pecifi-d target incu~2tion temp-rature- and to hold the ~ample ~ixtures at th-~e t~rget temp ratures for 20 the specifi-d incubation times Th- u-er interface code run by t~e CPU 20 di-plays, at all ~tages of th$~ plO~ , the current calculated ~ample liquid t mperature on th- di~play of terminal 16 Th- difficulty with di~playing an actual measured 25 ~ampl- t~mperature is that to mea-ur- the actual t-mperature of the reaction uixtur- requir-- in~ertion of a t-mperature mea-uring probe therein The ther~al ~a~ of the probe can signif$cantly alter the t~mperature of any w-ll in which it i~ placed ~ince the ~ampl- r-act$on ~ixture $n any 30 particular well i- oft-n only 100 uicrolit-r- in ~olum-Thus, the mere insertion of a t-mperatur- probe into a reaction mixtur- can cause a temperatur- gradient to ~xi-t between t~at reaction mixtur~ and neighboring ~ixtures Since the extra thermal mass of the temperatur- ~-n-or would 35 cause the reaction mixture in which it is i~n-r--d to lag be~ind in temperature from th- temperatures of th- reaction mixtures in other wells that have le-~ thermal ma~c errors can result in the amplification ~imply by att~mpting to measure the temperature Accordingly the instrument d-~crib~d h~rein calculates 5 the sample temperature from known factor- ~uch a- the block temperature history and the thermal time con~tant of the system and displays thi~ sample temperature on the display It has been found experimentally for the ~y~t-m de~cribed herein that if the ~ample tube~ ar- pro-~-d down into the 10 ~ample wells with at lea-t a minimum thr-~hold forc- F, then for the ~ize and ~hape of the ~ample tube~ u~ed in the preferred embodiment and th- ~ampl- volu~e- of approximately 100 microliters thermally driv~n conv-ction o~ c within the ~ample reaction mixture and the ~y~t~m act~ thermally 15 like a ~ingl- time constant, lin~ar ~y~tem Exp-riments have chown that ~ach ~ampl- tube mu~t b- p~-h~ down with approximately 50 grams of force for good well-wall-to-liguid thermal conductivity from well to well The h-ated platen design de~crib d below i- design-d to pu~h down on each 20 ~ample tube with about 100 gram- of force Thi~ ~inimum force ~y~bolized by force vector F in Figure 15 is necessAry to in~ur- that r-gardle-- of ~light diff-~nc-s in external dimen-ion~ as betw-en variou~ ~ample tube~ and variou- ~ample wells in th- ~ample block they all will be 25 pu~hed down with ~ufficient force to guarant-e the ~nug and flush fit for ~ach tube to guarant-- uniSorm thermal conductivity Any d-~ign which ha~ ~om- ~a~ple tubes with loos- fit~ in their C6~ G~ing ~ampl- ~ell~ and ~ome tubes ~ith tight fit~ will not b- abl- to achi-ve tight 30 temperature control for all tube~ b-cau~- of non-uniform thermal conductivity An in-uffici-nt l-v-l of force F
results in a temperatur- r-spon-- of the cample liguid to a step change in block t-mperatur- a~ ~ho~n at 286 in Figure 14 An adequate lev-l of force F r-sult~ in the t-mperature 35 response ~hown at 282 ~e result achi-v-d by the apparatus conctructed according to the teachings of the invention is that the temperature of each sample mixture behaves as if the sample is being well mixed physically during transitions to new temperatures. In fact, because of the convection currents caused in each sample mixture, the sample reaction mixture in each sample tube is being well mixed.
The surprising result is that the thermal behavior of the entire system is like an electrical RC circuit with a single time constant of 9 seconds which is about 1.44 times the half-life of the decay of the difference between the block temperature and the sample temperature. A GeneAmp* sample tube filled with 50 milliliters of sample has a time consta.nt of about 23 seconds.
In other words, during an upward change in temperature of the sample block, the temperature of the reaction mixture acts like the rise in voltage on the capacitor C in a series RC electrical circuit like that shown in Figure 16D in response to a step change in the voltage output of the voltage source V.
To illustrate these concepts, refer to Figure 14 which shows different temperature responses of the sample liquid to a step change in block temperature and to Figure 15 which shows a cross section through a sample well/sample tube combination. It has been found experimentally that when the volume of sample liquid 276 is approximately 100 microliters and the dimensions of the tube are such that the meniscus 278 is located below the top surface 280 of the sample block 12, and the force F pushing the sample tube into the sample well is at least 30 grams, the thermal time constant I (tau) of the system shown in Figure 15 is approximately nine seconds for a sample tube wall thickness in the conical section of 0.009 inches (dimension A). It has also been found experimentally that for these conditions, the thermal time constant I varies by about 1 second for every 0.001 inch change in wall thickness for the sample tube frustum (cone).
The thin-walled sample tubes described herein have been found to have thermal time constants of from about 5 to about 14 seconds when containing from 20 to 100 microliters of sample. Thicker tube walls result in longer time constants and more lag between a change in sample block temperature , .

and the r-sulting change in ~ample liquid t-mperatur-Mathematically, the expre~-ion for the thcroal response of the sample liquid temperature to a change in t-mperature of the sample block iB:
s (6) T,~ T(l-e' where T~, - the temperature of the ~~mpl- liguid ~T - th- temp-ratur~ differ-nc- b-tw--n the temp~ratur- of the ~ampl- block 12 ~nd the temperature of the ~ampl- liquid t - ~lap-ed time r ~ thermal time constant of the system, or the h-at capacity of sample d$vided by the th-rmal conductanc- from ~ampl- well wall to the sampl- liguid In Figure 14, the curve 282 represent~ thi- exponential temperature r-spons- to a th-or-tical st~p ch-~g~ in sample block t~mperature when th- force F pu-~ing down on the 20 sampl- tube i- sufficiently high The ~t-p change in temperature of the ~ample block i- shown aJ function 284, with rapid rise in temperature starting at time T1 Note how the t~mperatur- of the ~ampl- liquid exponentially increases in r-spon-e to the ~t-p chang- and a-ymptotically approaches 25 th- final ~a~pl- block t~mperatur~ A- u-ntion-d bri-fly abo~e, the curv- 286 r-pres-nts th- thermal .~ ~onse when th- ~r~l_ard ~-ating force F in rigur- lS is insufficient to cau-e a anug, flush fit betwe-n the cone of the sa~ple tube and the wall 290 of th- ~ample well Generally, th- thermal 30 re~ponse of curv- 286 will result if th- forc- F i~ s than 30 grams Note that although Figure 15 ~hows a small layer of air betw--n t~e cone of the ~ample tub- and the cample w-ll wall for clarity, this is ~xactly th- oppocite of the desir-d situation sinc- air is a good insulator and 35 would subetantially increa~e the thermal time constant of the sy~tem .~ .

The thermal time con-tant r i- analogou- to the RC time constant in a ~eries RC circuit where R corr--pond~ to the thermal r-sistance between the wall of the ~ample well and the sample liquid and C is the heat capacity of the ~ample 5 liguid Thermal r-si-tance is equal to the inverse of thermal conductance which is expr-fised in units w~tts-seconds per degree Relvin Because of the conv-ction currents 292 chown in the sample liguid in Figur- 15, everywh-r- in th- r-action 10 mixture the sample liquid i- at very n-arly the same temperature, and the flow of heat betw--n th- block and the sampl- is very nearly proportional to th- difference in temperature between the sample block and the sample reaction mixture The constant of proportionality is the thermal 15 conductanc- between the wall of th- ~a~ple well in the sampl- block 12 and the r-action ~ixtur- For different sample volumes or different tubes, i - , different wall thickn-s-e- or materials, th- th-roal tim- constant will be different In such a case, the us-r can ac part of his 20 specification of the PCR protocol ~nter the sample volume or tube type and th- ~achine will automatically look up the correct thermal tim- conetant for u-e in calculating the sample t~mperature In some embodiments, the user may enter the actual tim- constant, and the ~achin- will use it for 25 sampl- t-mp~ratur- t-mp-rature calculation To k--p th- th-rmal time constant as small as possible, the conical wall- Qf th- sampl- tub~s should be as thin as possibl- In th- pr-ferr-d embodiment, these conical walls are 0 009 inch-s thick wher-as th- walls of the cylindrical 30 portion of the ~ample tube ar- 0 030 inc~ thick The conical shape of the sample tube provide- a relatively large surface ar-a of contact wit~ th- Detal of the sample well wall in relation to the volume of the campl- ~ixture Molding of the ~ample tubes is done u-ing a ~cold 35 runner~ system and a four cavity mold such that four ~ample tubes are molded at each injection The molten plastic is injected at the tip of the cample tube cone ~o that any remnant of plastic will project into the cavity 291 between the tip of the ~ample tube and th- tip of th- ~ample well This prevents any remnant from interfering with the flush 5 fit between the tube and the well A maximum limit of 0 030 inches is placed on the size of any remnant plastic In various embodiments, 3 different grades of polypropylene each with differ-nt advantagec can be used The preferred polypropylene i- PD701 from Himont b-c-u~e it 10 is autoclavable However this pla-tic is difficult to mold because it has a low melt index This plastic has a melt index of 35 and a molecular density of 9 PD701 tcnds to leave flash and creates ~omewhat spotty guality partt but would work better if it was in~ected into the thick walled 15 part of th- mold instead of at th- tip of the conical section as i- currently done Generally, it is desirable to have a high melt index for ease of molding but also a high molecular density to maintain good strength and to prevent crazing or cracks under the thermal ~tre-s of the 20 autoclaving process at 260 F Another pla-tic, PPW 1780 from American Ho-scht has a melt index of 75 and a molecular density of 9 and is ~utoclav-ble Another pla-tic which may be used in som- ~mbodim~nt- i- Himont 444 This plastic is not autoclavabl- and needs to be ~teriliz-d in another 25 manner In alternative mbodiments, the tub~s may be molded using a ~hot runner" or ~hot nozzl-~ ~y-t m ~here the temperature of the molt-n plastic 1- cor~oll-d right up to the gate of the rold al-o, in ~om- e~bodim-nt~, multiple 30 gates may be used Hov-ver, n-ith-r of thes- t-chnigues ha-been experimentally proven at the time of filing to be better than the currently used ~cold runner~ ~ystem The faet that thc ~y-t-m acts thermally l$ke a single time constant RC circuit is an important result, because it 35 means that if the thermal conductance from the sample block to the ~ample reaction mixture is knovn and uniform, t~e thermal response of the ~ample mixtures will be known and uniform Since the heat capacity of the sample reaction mixture is known and constant, the temperature of the ~ample reaction mixture can be computed accurately using only the S measured history of the block temperature over time This eliminates the need to measure the ~ample temperature thereby eliminating the errors and mechanical difficulty of putting a probe with nonnegligible thermal ma6s into a ~ample well to measure th- ~ampl- t-mp-ratur- dir-ctly 10 thereby changing the thermal ma~ of the sample in the probed well The algorithm which mak-s thi- calculation model~ the thermal behavior of the system after a ~ingle time constant series R-C electrical circuit Thi- model u-e~ the ratio of 15 the h-at capacity of the liquid ~ampl- divided by the thermal conductance from the sa_pl- block to the sample reaction mixture The heat capacity of the cample reaction mixture is equal to the ~pecific heat of the liquid times the mass of the liquid The thermal re~i~tance i~ ~qual to 20 one over the thermal conductance from the sample block to the liquid reaction mixture through the ~ampl- tube walls When thi~ ratio of heat capacity divided by thermal conductance i~ expre~ed in con~i-tent units, it ha~ the dimension of tim- For a fixed sample volume and a fixed 25 sample compocition both of which ar- the s~ce in every ~ample well and a fixed thermal conductance, the ratio is also a constant for every sample well, and i~ called the thermal ti~e con-tant of the sy-tem It is the time r-quired for the ~ample t-mperature to come within 36 8~ of 30 the block temperatur- after a sudd-n step change in the block temperature There is a ~athematical theorem used in the analy~is of electronic circuits that hold- that it i~ po~-ible to calculate the output response of a filter or other linear 35 system if one knows the impul~e response of the ~y~tem This impulse response is al-o known a6 the transfer function In the ca~e of a ~-ries RC circuit, the impulse response is an exponential function as ~hown in Figure 16A
The impulse stimulu~ resulting in the response of Figure 16A
is as ~hown in Figure 16B The ~athematical theorem 5 referred to above holds that the output response of such a linear system can be determined by calculating the convolution of the input ~ignal and a weighting function where the weighting function is the impulse response of the system reversed in time Th- convolution i- otherwise known 10 as a running weighted average although a convolution is a concept in calculus with infinitely ~mall ~tep ~iz-- whereas a running weighted average ha- di-cre-t ~t-p ~izes, i - , multiple samples The impul-e response of the series ~C
circuit ~hown in Figure 16D as such that when the voltage of 15 the voltage generator V suddenly ri--s and falls with a spike of voltage as shown in Figur- 16B, the voltage on the capacitor C suddenly rises to a peak at 294 in Figure 16A
which i- equal to the peak voltage of the impulse shown in Figure 16B and then exponentially d-cayc back to the steady 20 state voltage V~ Th- r-sulting weighting function i6 the impulse respon-e of Figure 16A turned around in time as shown in Figure 16C at 385 Superimposed upon Figure 16C is a hypothetical curve 387 illustrating a typical temperature history for the 25 temperature of th- ~ample block 12 for an approximate ~tep change in temperature Also shown ~uperimpos-d upon Figure 16C are the times of five temperature sample periods labelled T1 through T5 According to the t-achings of the invention, the sample temperature is calculated by 30 multiplying the t-mperatur- at ~ach one of the-e times T~
through T5 by the ~alue of the weighting function at that particular time and then ~umming all these products and dividing by 5 The fact that the thermal ~ystem acts like a single time constant linear circuit is a ~urprising re~ult 35 based upon the complexities of thermal heat transfer considerations for this complicated thermal system In one ~mbodiment, the calculation of the sample temperature i6 adjueted by a ~hort delay to account for transport lag caused by differ-nt thermal path lengths to the block temperature ~en-or and the ~arple liguid The 5 calculated sample temperature is displayed for the user's information on the terminal 16 ~hown in Figure 1 Figure 17 6hows the temperature response results for six different wells ~pread throughout the 96 well ~ample block for a otep change in sample block t-mperature from a 10 relatively lower temperatur- in the hybridizat$on/exten~ion temperature range to the relativ-ly higher te~perature of approximately 94 C u-ed for denaturation Th- graph of Figure 17 ~hows good agree~ent betw-en the predicted expon-ntial ri-e in sample t~mperature if th- sy-tem were 15 perfectly analogous to the ~eries RC circuit chown in Figure 16D, and al-o ~hows ~xc-ll-nt uniformity of te~perature response in that the temperatur-s of the ~ix ~a~ple wells u~ed for this ~tudy a~ymptotically ~ettle in at temperatures very cloee to ~ach other and in a denaturation temperature 20 ~tolerance~ band which i~ approximately 0 5 C wide In one ~mbodiment, the ten most recent block temperature ~amples are us-d for the ~unning weighted average, but in other embodiments a different number of tempersature hi~tory ~amples may be u-ed The good 25 agreement ~ith theoretically pr-dict-d re-ult~ ~tem~ from th- fact that the th-rual convection curr-nt~ mak- the ~ampl- liquid~ w-ll mix-d thereby cau-ing th- ~y-tem to act in a linear fa-hion The uniformity between ~ampl- temp-ratur-~ in ~arious 30 ~ample wells apread throughout the 96 w-ll array re~ults from dynamic and static local balance and local ~ymmetry in the ~ample block ~tructure a~ well as all the other thermal design factors detailed herein Not- ho~-ver that during rapid temperature changes all the ~ampl- well~ will have 35 temperatures within O 5 C of each other only if the u~er has carefully loaded each eample well ~ith the ~ame mass of sample liquid Ineguality Or ~ass in different well~ does not cause unequal te~peratures in ~teady stat-, unchanging conditions, only during rapid chang-s The ~as6 of the sample liquid in ~ach well i- the dominant factor in s determining the heat capacity of ~ach ~ample and, therefore, is the dominant factor in the thermal time constant for that particular sample well Note that the ability to cauce the sample liguid in all the sample wells to cycle up and down in t~mperature in 10 unison and to stabilize at target t mp-ratur-- very near each other, i . 8 ., in tolerance bands that ar- only 0 5 C
wide, also dependc upon the forc- F in Figur- 15 This force must exce-d a minimum thre6hold force before the thermal time constants of all sample well- load-d with 15 similar ma~se~ of sample liguid will have the ~ame time constant Thi- minimum threchold force has been experimentally det-rmined to be 30 grams for th- ~ample tube and sampl- well configuration describ d her-in For higher levels of accuracy, the minimum thr--hold forc- F in Figure 20 15 should be establi-hed at at l-ast 50 grams and preferably 100 grams for an additional margin of safety a- not-d abov-The importance of thermal uniformity in ~ample welltemperature can be appreciated by r-fer-nce to Figure 18 ~ his figure shows the relation-hip between the amount of DNA
25 generated in a PCR cyclc and th- actual ~ampl- tamperature during the denaturation interval for on- instance of amplification of a certain s-gment of DNA The slope of function 298 b tw-en temperatur-s 93 and 9S degrees centigrade is approximately 8% per d-gr-e centigrade for 30 this particular cegment of DNA and pri~ers Figure 18 shows the general shape of the curv- wbich relates the ~mount of DNA generated by amplification, but the details of the sbape of the curve vary witb ~very different cas- of primers and DNA target Temperatures for denaturation abov- 97 d-grees 3s centigrade are gencrally too bot and result in decr-asing amplification for increasing denaturation temperature Temperatures between 95 and 97 degr-e- c-ntigrade are generally just right Figure 18 illu-trates that any ~ampl- well containing this particular DNA target ~nd prim~r combination which 5 stabilizes at a denaturation temperature of approximately 93~C is likely to have 8% le~6 DNA generated over the course of a typical PCR protocol than wells denatured at 94~C
Likewise, sample liquids of this mixture that stabilize at denaturation temperatures of 95 C are likely to ~av- 8% more 10 DNA generated therein than is gen-rated in sample wells which ~tabilize at denaturation temperatur-s of 94'C
Because all curves of this nature have th- same general shape, it is important to have uniformity in sample temperature The ~ample temperatur-s calculated as de~crib d above are used by the control algorit~m for controlling the heater~ and flow through the ra~p cooling ~h~n~els and to determine how long the sample- ~av- been ~-ld at various target temperatures The control algorithm u-e~ the~e times 20 for comparison with the desired time~ for each incubation period as entered by the user When tbe timefi match, the control algorithm takes the appropriate ct-p~ to h-at or cool the sampl- block tov~rd the targ-t temperature defined by the u~er for the next incub~tion When the calculat-d ~ampl- te~perature i- vithin one degr-- centigrade of the ~-tpoint, i - , the incubation te~pQrature programmed by th- user, the control program cause~ a timer to start This timer ~ay b pr--et to count down from a number set ~o a~ to time out the interval 30 ~pecified by th- user for th- incubation being performed The timer ~tarts to count down from the pr-s-t count when the calculated sample temperature is wit~in one degree centigrade When tbe timer reaches a zero count, a signal is activated which cau--s the CPU to tak- action~ to 35 implement the next ~egment of the PCR protocol Any way to time the specified inter~al will cuffice for purposes of practicing th- invention Typically, the tolerance band around any particular target temper~ture is plu8 or minu- 0 5 C once the target temperature is reached, the computer hold~ th- ~a~ple block 5 at the target temperature using the bias cooling channels and the film heater such that all the ~amples remain clo~e to the target temperature for the ~pecified interval For the thermal ~y~tem described h-rein to work well, the thermal conductance from the ~ample block to oach ~ample 10 must be known and uniform to vithin a very clo~e tolerance Otherwi~e, not all ~amples will be h-ld within th- ~pecified toleranc- band of the target temperature when the timer starts and, not all the sampl~s vill experience the ~ame incubation intervals at the target temperature Also, for thi- thermal ~ystem to work well, all fample tubes must be i~olated from variables in the ambient environment That i-, it i~ unde-irable for ~ome sample tubes to be cooled by drafts while other ~ample tubes in different phy-ical positions do not ~xp-rience the ~ame 20 cooling effects For good uniformity it i~ highly desirable that the temperaturef of all the ~ampl-s be determined by th~ temperature of the ~ample block and by nothing else Isolation of th- tube- from the ambient, and application of the minimum thr~shold force F p~ ing down on 25 the ~_mple tube~ i- achi-v-d by a heated cov-r over the cample tubes and ~ample block Ev~n though the ~ampl- liguid i- in a ~ample tube pr~-s-d tightly into a t~mp-r-tu~ cG troll-d D-tal block, tightly capped, with a meniacu~ w-ll below the ~urface of 30 the temperature cGn~olled metal block, th- ~amples ~till lose their heat upward by convection Significantly, when the ~ample is very hot (the d-naturation temperature is typically near the boiling point of the ~ampl- liquid), the sample liquid can lose a very ~ignificant amount of h-at by 35 refluxing of water vapor In this proces6, water evaporates from the ~urface of the hot ~ample liquid and condenses o~

the inner walls of the cap and the cooler upper parts of the sample tube above the top surface of the ~ample block If there i~ a relatively large volume of ~ample, conden-ation continues, and condensate builds up and run- bac~ down the 5 walls of the sample tube into the reaction mixture This "refluxing" process carries about 2300 joules of heat per gram of w~ter refluxed This proces6 can cause a drop of several degrees in the surface temperature of a 100 microliter reaction ~ixture th-r-by cau~ing a large 10 reduction of efficiency of the r-action If the reaction mixture i~ ~mall, ~ay 20 microliter~, and the sample tube has a relativ-ly large ~urface area above the top surface of the ~ample block, a significant fraction of the water in the reaction mixture may evaporate 15 Thi- water may then condens- in-id- the upper part of th-sample tube and remain there by ~urface ten~ion during the remainder of the high temperatur- part of the cycle This can so concentrate the remaining r-action mixture that the r-action i- impaired or fails completely In the prior art PCR thermal cyclers, thi~ refluxing problem was dealt with by ov-rlaying th- reaction mixture with a layer of oil or melted wax This $mmi~cible layer of oil or wax floated on the aqu-ou~ reaction mixture and prevented rapid evaporation However, labor was r-guired to 25 add th- oil vhich rai-ed pr.~ ing co~t~ Further, the pr~-enc~ of oil interf-red with later ~t-ps of processing ~nd analy~i~ and cr-ated a po~ibility of contamination of the ~a~ple In fact, it is known that indu-trial grade mineral oils have in the pa~t contaminat-d ~ampl-s by the 30 unknown presenc- of contaminating factor~ in the oll which were unknown to the users The need for an oil overlay i~ eliminated, and the problems of heat loss and concentration of the reaction mixture by evaporation and unp~ ~ictable theroal effects 35 caused by refluxing are avoided according to the teachings of the invention by enclosing the volume above the sample .

block into which the upper part~ of the sample tubes project and by heating this volume from above by a heated cover sometimes hereafter also called the platen Referring to Figure 19, there is ehown a cross 5 sectional view of the ~tructure which is used to enclose the sample tubes and apply downward force thereto so as to supply the minimum threshold force F in Figure lS A heated platen 14 is coupled to a lead ~crew 312 ~o as to move up and down along the axi- ~ymbolized by arro~ 314 ~ith 10 rotation of the lead ~crew 312 The lead ~crew i- threaded through an opening in a sliding cover 316 and is turned by a knob 318 The platen 314 i8 heated to a temperature above the boiling point of water by resi~tance heaters (not shown) controlled by computer 20 Th- ~liding cov-r 316 elid-s back and forth along the Y axi6 on rail- 320 and 322 The cover 316 -includes vertical sides 317 and 319 and also includes vertical sides parallel to the X-Z plane (not ~ho~n) which enclose the tample block 12 and ~ample tubes Thi- ~tructure 20 substantially prevent drafts from acting on the sample tubes of which tubes 324 and 326 are typical Figure 20 i- a pe~s~e_~ive view of the ~liding cover 316 and ~ample block 12 with the sliding cover in retracted position to allow access to the eample block The ~liding 25 cover 316 re--mbl-~ th- lid of a r-ctangular box with vertical wall 328 having a portion 330 removed to allow the sliding cover 316 to ~lide over the ~ample block 12 The ~liding cover i~ ~oved along the Y axi~ in Figure 20 until tbe cover i8 centered over the ~ample block 12 The user 30 then turns the knob 318 in a dir-ction to lower the heated platen 14 until a mark 332 on th- knob 318 lines up with a mark 334 on an e~cutcheon plate 336 In some embodiments, the escutch-on plate 336 ~ay b- p-rcanently affix-d to the top ~urface of the sliding cover 316 In other embodiment6, 35 the eseutcheon 336 ~ay be rotatable ~uch that the index mark 334 may be placed in different positions ~hen different size CA 022660l0 l999-04-Ol sample tubes are used In oth-r words, if tall-r cample tubes ar- used, the heated platen 14 n--d not be lowered a5 much to apply the minimum thre-hold force F in Figure 15 In use, the user screws the scr-w 318 to lower the platen 14 5 until the index marks line up The u~er then knows that the minimum threshold force F will have been applied to each sample tube Referring jointly to Figur-- 15 and 19, prior to lowering the heated platen 14 in Figur- 19, th- plastic cap 10 338 for ~ach sampl~ tube ~tick- up about O S millimetcrs Above the level of the top of the wall- of a plastic tray 340 (Figure 19) which holds all the ~ampl- tubes in a loose 8x12 array on 9 millimeter centers Th- array of ~ample wells can hold up to 96 ~icroAmp~ PCR tub-s of 100 ~L
15 capacity or 48 larger Gene~mp~ tub-s of 0 5 ml capacity The details of this tray will be ~i~c~ in gr-ater detail below The tray 340 has a planar surface having an 8x12 array of holes for sample tubc- This planar ~urface is ~hown in Figures 15 and 19 as a horizontal line which 20 intersects the 6ample tubes 324 and 326 in Figure 19 Tray 340 also has four v-rtical wall- two of vhich are ~hown at 342-and 344 in Figure 19 The top l-vel of the-e vertical walls, shown at 346 in Figure 15, ~ctablishes a rectangular box which defines a reference plane As best ~een in Figure 15, the capc 338 for all the sample tubes pro~ect above this referenc- plane 346 by some small ~mount which is designed to allow th- caps 338 to be soften-d and deform-d by the he~t-d platen 14 ~nd ~quashed"
down to the level of the reference planc 346 In the 30 preferred embodiment, the heated platen 14 i- kept at a temperature of 105~C by the CPU 20 in Figure 1 and the bus 22 coupled to resistance heaters (not ~hown) in the platen 14 In the preferred embodiment, the knob 3~8 in Figure 19 and the lead screw 312 are turn-d until the heated platen 14 35 descends to and makes contact with the topc of the caps 338 In the preferred embodiment, the caps 338 for the cample CA 022660l0 l999-04-Ol tube- ar- mad- of polypropylene . Th--- cap- ~often ~hortly after they come into contact with the heated platen 14. As the caps soften, tbey deform, but they do not lose all of their elasticity After contacting the caps, tbe heated 5 platen is lowered further until it rests upon tbe reference plane 346. This further lowering deforms the caps 338 and causes a minimum threshold force F of at least 50 grams to push down on ~ach ~ample tube to k-ep ~ach tub- w-ll aeated firmly in its ~ample well The amount by whicb th- caps 338 lO proj-ct above the referenc- plan- 346, and th- amount of deformation and residual elasticity when the h-at-d platen 14 rests upon the reference planc 346 is designed such tbat a minimum threshold force F of at l-ast 50 grams and preferably lO0 grams will have been achiev-d for all ~ample 15 tubes then present after tbe h-at-d plat-n 14 bas d~s-en~ed to the level of tbe refer-ncc plane 346.
The heated platen 14 and tbe four vertical walls and planar surface of tbe tray 340 form a heatcd, ~ealed compartment when the platen 14 i- in contact with tbe top 20 edge 346 of the tray The plastic of the tray 340 has a relatively poor ther~al conductivity property It has been found experimentally that contacting tbe h-at-d platen 14 with thc caps 338 and tbe isolation of tbe portion of the sample tubes 288 which project above the top level 280 of 25 tbe ~a~pl- block 12 by a wall of ~aterial which has relatively poor thermal conductivity has a beneficial result With tbis~structure, tbe ~ntire upper part of the tub~ and cap ar- ~ou~t to a temperature which $- high enough that littl- or no conden~ation for~- on tbe inside 30 surfaces of the tube and cap ~ince the heated platcn i- kept at a temperature abcve the boiling point of water Thi~ is true even when the sample liquid 276 in Figure 15 ic heated to a tcmperature near its boiling point Thi~ ~liminates the need for a layer of i~niscibl- material ~uch as oil or 35 wax floating on top of the ~ample mixture 276 thereby reducing the amount of labor involved in a PCR r-action and eliminating one ~ource of poseible contamination of the sample It has been found experimentally that in ~pite of the very high temperature of the heated cover and ite close 5 proximity to the s~mple block 12, there is little affect on the ability of the sample block 12 to cycle accurately ~nd rapidly between high and low temperatures The heated platen 14 prevcnts cooling of th- ~amples by the refluxing process noted earlier b cause it keeps the lo temperature of the caps above the condensation point of water thereby k-eping the insides of the caps dry This also prevents the formation of aerosol~ when the caps are removed from the tubes In alternative embodiments, any ~-ans by which the 15 minimum acceptabl- downward forc- F in Figure 15 can be applied to each individual sample tube regardl-ss of the number of sample tubes Pr-eent and which will prevent condensation and refluxing and convection cooling will suffice for purpoces of practicing the invention The 20 application of this downward force F and the use of heat to prevent refluxing and undecir-d sampl- liquid concentration need not be both impl-m-nt-d by the ~ame ~ystem as is done in the preferr-d mbodiment The sample tub s may vary by a few thousandths of an 25 inch in their overall h-ight Further, the caps for the sample tube- may also vary in height by a few thousandths of an inch Also, each conical sample wcll in the ~ample block 12 ~ay not b~ drill-d to ~xactly thc ~am- d-pth, and ~ach conical sample well in the ~ample block may be drilled to a 30 slightly differcnt diamctcr and angle ~hus, when a population of capped tubee i- plac-d in the ~amplc block so as to be seated in the correeponding sample well, the tops of the caps will not all nece-~rily be at the same height The worst case discrepancy for this height could bc as much 35 as 0 5 millimeters from the highest to the lowest tubes If a perfectly flat unheated platen 14 mounted so that it is free to find its own position were to be pressed down on such an array of caps, it would first touch th- three tallest tubes As further pressure was applied and the tallest tubes were compressed ~omewhat, the plat-n would S begin to touch some caps of lower tubes There is a distinct possibility that unles~ the tube and cap assemblies were compliant, the tallest tubes would be damaged before the ~hortest tubes were contacted at all Alternatively, the force ncces~ry to compre~s all th- tall tubes 10 sufficiently 80 as to contact th- ~hort--t tube could be too large for the device to apply In eith-r ca~e, one or more short tubes might not be pressed down at all or might be pressed down with an insufficient amount of force to guarantee that th- thermal time constant for that tube was 15 equal to tbe thermal time constants for all the otber tubes This would result in the failure to achieve the ~ame PCR
cycle for all tubes in the ~ample block ~ince some tubes with different thermal time con6tants woulc not be in step with the other tube~ Heating the platen and softening the 20 caps eliminates these risks by eliminating the manufacturing tolerance errors which lead to differing tube heights as a factor In an alternative ~mbodiment, th- entire hcated platen 14 is cover-d with a compliant rubber layer A compliant 25 rubber layer on the h-ated platen would solve the height tolerance problem, but would al-o act as a thermal insulation layer which would d-lay th- ~low of h-at from the heated platen to the tube caps Further, with long use at high temperatures, most rubb r material~ deteriorate or 30 become hard It $~ therefore d-sirable that the heated platen ~urface be a metal and a good conductor of heat In another alternative embodiment, 96 individual springs could be mounted on the platen ~o that ~ach spring individually presses down on a single ~ample tube This is 35 a complex and costly ~olution, bowever, and it requires that the platen be aligned over the tube array wieh a ~echanical precision which would be difficult or bothersome to achieve The necessary individual compliance for each sample tube in the preferred embodiment is supplied by the use of plastic caps which coll~p~e in a predictable way under the 5 force from the platen but which, even when collapsed, ~till exert a downward force F on the sample tubes which is adequate to keep each sample tube seated firmly in its well In the sample tube cap 338 ~hown in Figure 15, the surface 350 should be free of nicks,-fla-h and cuts so that 10 it can provide a hermetic ~eal with the $nner vall- 352 of the sample tube'288 In the pr-ferr-d embodiment, the material for the cap is polypropylene A suitable material might be Valtec*HN-444 or PD701 polypropylene manufactured by Himont as described above or PPW 1780 by American 15 Hoescht In the preferred embodiment, the wall thickness for the domed portion of the cap i~ 0 130 + 000 - 0 005 inches The thickness of the ~boulder portion 356 is 0 025 inches and the width of the domed ~haped portion of the cap is 0 203 inches in t~e preferred embodiment Any material and configuration for the caps which will cause the minimum thre~hold force F in Figure lS to be applied to all the sa~ple tu~e~ and which will allow the cap and upper portions of the sample tubes to be heated to a temperature high enough to prevent condenF~tion and 25 refluxing ~ill ~uffice for purpose~ of practicing the invcntion The dome -'h~ cap 338 has a thin wall to aid in deformation of the cap Becaw e the heated platen is kept at a high temp~rature, th- wall thirkr-~ of the domed sh~pe~ cap can be thicX enough to be ~asily manufactured by 30 injection molding ~ince the necessary compli~nce to account for differences in tube height is not necessary ~t room temperature The platen can be kept at a temperature anywhere from 94 C to l~O C according to the teachings of the invention 35 although the range from lOO C to llO C i~ preferred to prevent refluxing ~ince the boiling point of water is lOO C

* Trade-mark In this temperature range, it has been ~xperimentally found that the caps soften just enough to collapse ea~ily by as much as 1 millimeter Studies have ~hown that the elastic properties of the polypropylene u~ed are ~uch that even at 5 these temperatures, the collapse is not entirely inelastic That is, even though the heated platen causes permanent deformation of the caps, the material of the caps ~till retain a significant enough fraction of their room temperature elastic modulus that th- minimum thre~hold force 10 F is applied to each ~ample tube Further, th- h-ated platen levels all the caps that it contacts without excessive force r-gardle~s of how many tubo- are pre~ent in the sample block because of th- ~oft-ning of the cap Because the cap temperature i~ abov- the boiling point 15 of water during the entire PCR cycle, the inside surfaces of each cap remain completely dry Thu-, at the ~nd of a PCR
process, if the samples are cooled to room temperature before being removed from the sampl- block, if the caps on each sample tube are opened, ther- i~ no po~sibility of 20 creating an aerosol ~pray of the ~ample tube contents which could result in cross contamination Thi~ i8 because there is no liquid at the cap to tube seal when the 6eal is broken This i- extremely advantageou~, becau~e tiny particles 25 of aero~ol containing amplified product DNA can contaminate a laboratory and get into ~ample tub-- containing ~amples from other ~ourc~ q , other pati-nt~, thereby poc~ibly causing fal~e pocitive or negative diagno~tic re~ult~ which can be very troublesome User- of the PCR amplification 30 process are extremely concerned that no aero601s that can contaminate other ~amples be created A ~ystem of disposable plastic items iB u~ed to convert the individual sample tubes to an 8x12 array which is compatible with microtiter plate fornat lab ~quipment but 3s which maintains sufficient individual freedom of movement to compensate for differences in the various rates of thermal expansion of the system components The relationship of the therm~lly compliant cap to th- r--t of thic ~y-t-m ie best seen in Figur- 21A which is a cro-s ~ectional view of the sample block, and two ~ample tube- with caps in place with 5 the sample tubes being held $n place by the combination of one embodiment of a plastic 96 well microtiter tray and a retainer Figure 21~ is an alternative, preferred e~bodiment showing the structure and interaction of most of the various plastic disposable it-ms of the system The 10 rectangular plastic 96 well microtiter plate tray 342 rests on the surface of the sample block 12; The top edge 346 of the frame 342 has a height ~hich i- approximately 0 5 millimeters ~horter than the height of th- caps of which cap 364 is exemplary All of the capped tubes will project lS higher than the ~dge 346 of the frame 342 The frame 342 is configured such that a downward extending ridge 366 extends into the guardband groove 78 through it- entire length The frame 342 does however have a gap (not shown) which corresponds to the gap in the groove 78 for the temperature 20 sensor 6hown in Figure 2 in plan view and in Figure 7 in cross-sectional view The refer-nce plane 346 mentioned above is established by the top of the frame 342 How this reference plane interacts with the heated platen is as follows Prior to 25 screwing down the knob 318 in Figure 20 to line up the index marks 332 and 334 to start an amplification run, a calibration proces~ will have be-n performed to locate the position of th- index mark on the e-cutc~on platen 336 in Figure 20 Thi- calibration i~ ctart-d by placing the frame 30 342 in Figure 21 in position on the ~ampl~ block The frame 342 will be empty however or any sample tubes therein will not have any caps in place Then, the knob 318 is screwed down until the heated platen 14 is firmly in contact with the top edge 346 of the frame 342 around its entire 35 parameter When the knob 318 has been ~cr-wed down sufficiently to allow the heated platen to reast on the reference plane 346 and to press the frame 342 firmly against the top surface 280 of the ~ample block, the rotatable escutcheon 336 of the preferr-d embodim-nt will be rotated until the index mark 334 on the e~cutcheon lines up 5 with the index mark 332 on the knob 318. Then, the knob 318 is rotated counterclockwise to raice the platen 14 and the cover 316 in Figure 19 is slid in the negative Y direction to uncover the frame 342 and the ~ample block 12. Sa~ple tubes with cap~ loadQd with a ~ampl- ~ixtur- ~ay then be 10 placed in position in the frame 342. The heated cover 316 is then placed back over th- ~ample block, and the knob 318 is turned clockwise to lower the heated platen 14 until the index mark 332 on the knob lines up with the index mark 334 as previously positioned This guarantees that all tubes 15 have been fir~ly ~eated with the ~inimum force F applied The use of the index mark~ give~ the u~er a- ~imple, verifiable task to perform If there are only a few sample tube~ in place, it will take only a ~mall amount of torqu- to lin- up the index 20 marks 332 and 334. If there are ~any tubes, however, it will take more torque on the knob 318 to linc up the index marks This is becau~e ~ach tube is resi~ting the downward movement of the heated platen 14 a~ the cap~ deform However, the u~er is as~ured that when the index marks 33 2 25 and 334 are aligned, the heated platen will once again be tightly plac-d against the top ~dge 346 of th- frame 342 and all tub-s will have the ~inimum thre~hold force F applied thereto Thi~ virtually guarant-e~ that the thermal time constant for all the tubes will be ~ubstantially the ~ame ~n alternative embodiment-, the index mark~ 332 and 334 may be dispensed with, and the knob 318 may ~i~ply be turned clockwi~e until it will not turn any ~ore This condition will occur when the heated platen 314 ha- reached the top edge or reference plane 346 and the pla~tic frame 342 has 35 stopped further downward movement of the heated platen 14 Obviously in this alternative embodiment, and preferably in the index mark embodiment described above, the pla~tic of the frame 342 will hav- a melting temperature which is sufficiently high to prevent deformation of the plastic of the frame 342 when it i~ in contact with the heated pl~ten 5 14 In the preferred embodiment, the plastic of the frame 342 is celanese nylon 1503 with a wall thickness of 0 05 inches An advantage of the abov- de6cribed ~y~tem i~ that sample tubes of different height~ may be u-ed ~imply by 10 using frames 342 having different heights Th- frame 342 should have a height which i- approximately 0 5 millimet~rs shorter than the plane of the tip~ of the capped tubes when both are seated in the ~ampl- block In the preferr-d embodiment, two different tube heights are u~ed The range 15 of motion of the lead ~crew 312 which drives the heated platen 14 in Figure 19 must be cufficient for all the different sizes of ~ample tubes to be u~ed Of course, during any particular PCR proce~6ing cycle, all tubes must be the ~ame height The ~ystem described above provides uniform temperatures in the ~ample block, uniform ther~al conductance from block to ~ample, and isolation of the sample tubes from the vagaries of the ambient environment Any number of ~ample tube~ up to 96 may be arrayed in the 25 microtiter plate format The ay~tem allows accur~te temperature control for a very large num~er of ~amples and a vi~ual indication of the ~ample temp-rature~ for all samples ~ithout actually mea~uring the temperature of any sample As the container for PCR r-actions, it has been common in the prior art to use polypropylene tubes which were originally designed for microcentrifuge~ Thi~ prior art tube had a cylindrical cros----ction closed at the top by a ~nap-on cap which makes a gac-tight ~eal Thi~ prior art 35 tube had a bottom ~ection which compri~ed the frustrum of a cone with an included angle of approximately 1~ degrees When euch a conical sample tube ig pressed down into a sample well of a sample block with a conical cavity with the same included angle, and when the ~ample mixture in the tube lies entirely within the conical volume and b-low the top 5 surface of the sample block, the thermal conductance between the block and the liquid can be made adeguately predictable for good uniformity of sample temperature throughout the array To achieve adeguate control of the thermal conductance between the sample block and th- sampl- mixture, 10 the includ-d angles of tbe conical tube and th- ~ample well must match closely, and the conical cu-rfaces of the tube and well must be ~mooth and held together in flush relation Further, the minimum threshold force F must be applied to each sample tube to press each tube tightly $nto the ~ample 15 well ~o that it doe~ not rise up or loosen in the well for any reason during thermal cycling, such a~ steam formation from trapped liquid in space 291 in Figure 15 Finally, each tube must be loaded wit~ the same amount of sample liquid If the above licted conditions are met, the thermal 20 conductance between the sample block and the ~ample liquid in each tube will be pr-dominantly determined by the conductance of the conical plastic wall 368 in Figure 15 and a boundary layer, (not shown) of the sample liquid at the inside surfac~ 370 of the conical sample tub- wall The thermal conductance of the plastic tube walls is determined by their thickn---, which can be closely controlled by the in~ection molding method of manufacture of the tubes The sample liguid in all th- sample tubes has virtually identical thermal properti--It has been found by experiment and by calculation that a molded, one-piece, 96-well microtiter plate ic only marginally feasible for PC~ becau~e the differences in the thermal expan~ion coefficients between aluminum and plastic lead to dimensional changes which can de~troy the uniformity 35 of thermal conductance to the sample liguid acrots the array That is, since each well in ~uch a one-piece plate is connected to each other well through the ~urface of the plate, the dictances between the well- ar- d-termined at the time of initial manufacture of the plate but change with changing temperature eince the plastic of the plate has a 5 significant coefficient of thermal expansion Also, distances between the cample wells in the metal eample block 12 are dependent upon the temperature of the eample block since aluminum aleo has a significant co-fficient of thermal expansion which ie different than that of plaetic To have 10 good thermal conductance, each eample well in a one-piece 96-well microtiter plate would have to fit almoet perfectly in the corresponding well in the ~ample block at all temperatures Since the temperature of the sample block changes over a very wide range of temperatures, the 15 distances between the sample welle in the ~ample block vary cyclically during the PCR cycl- Becau-- th- coefficients of thermal expansion for plastic and aluminum are sub-tantially different, the dietance6 of the well separation in the sample block would ~ary differently over 20 changing temperatures than would the dietances between the sample wells of a plastic, one-piece, 96-well microtiter plate Thus, as an important criteria for a perfect fit between a eample tube and the corresponding eample well over 25 the PCR temperature range, it i- n~ ry t~at each sample tube in th- 96-well array be individually free to move laterally and each tube mu-t be individually free to be pre-sed down vertically by whatever amount ie necessary to make flush contact with the wall- of the sample well The eample tubes ueed in th- invention are different from the prior art ~icrocentrifuge tubee in that the wall thickness of the conical fruetrum poeition of the eample tube is much thinner to allow faeter heat transfer to and from the eample liquid The upper part of these tubes has 35 a thicker wall thickness than the conical part In Figure 15, the wall thickness in the cylindrical part 288 in Figure 15 is generally 0 030 inches while the wall thickness for t~e conical wall 368 is 0 009 inches Because thin parts cool faster than thick parts in the injection molding process, it is important to get the mold full before the 5 thin parts cool off The material of the sample tubes must be compatible chemically with the PCR reaction Glass is not a PCR
compatible material, because DNA ctickc to gla~c and will not come off which would interfere with PCR aoplification 10 Preferably an autoclavable polypropylene i- u~ed Three types of suitable polypropylene were identifi-d earlier herein Some plastics are not compatible with the-PCR
process because of outgassing of material~ from the plastic or because DNA sticXs to the plastic wall- Polypropylene 15 is the best known class of pla-tic~ at thi- time Conv-ntional injection molding t~chnigu-s and mold manufacture techniques for the injection mold will suffice for purposes of practicing the invention The use of cone shaped ~ample tubes translates 20 substantially all manufacturing toleranc- errorc to height errors, i e , a variance from tube to tube in the height of the tip of the cap to the top of th- ~ampl- block when the sample tube is ceated in the ~ample well For example, an angle error for the angle of the aample tube walls is 25 convert-d to a height error when th- tube ic plac-d in the sample block becauce of the micmatch b tw-en th- tube wall angle and the sample well wall angl- Likewi-e, a diameter error in the dimensionc of the cone would alco tranclate into a height error cince the conical part of the tube would 30 either penetrate deeper or not a- ~uch a- a properly dimensional tube For good uniformity of thermal conductanc- acrocs the array, a good fit between the ~ampl- tub-- and th- cample well must exict for all 96-wells over the full temperature 35 range of o to lOO C regardless of differences in thermal expansion rates Also, each of the 96 cample tubes must have walls with dimensions and wall thicknesses which are uniform to a very high degree Each ~ample tube in which sample mixturc is to be held should be fitt-d with a removable gas-tight cap that makes a ga6-tight ~eal to S prevent loss of water vapor from the reaction mixture when this mixture is at or near its boiling point 6uch that the volume of the ~ample mixture does not decrease All these factors combine to make a one-piece microtiter plate with 96 individual ~ample well6 extremely difficult to ~anufacture 10 in a manner so as to achieve uniform thermal conductance for all 96 wells Any ~tructure which provides the necescary individual lateral and vertical degrees of freedom for each ~ample tube will ~uffice for purposes of practicing the invention Aceording to the teaching6 of the preferr-d embodiment of the invention, all the above not-d r-guirement6 have been met by using a 4 piece disposable plastic ~y~tem This system gives ~ach ~ample tube ~ufficient freedom of motion in all necessary directions to compensate for differing 20 rates of thermal ~xpan-ion and yet retains up to 96 ~ample tubes in a 96 well microtitcr plate format for user convenience and compatibility with other laboratory eguipment which i~ siz-d to work with the indu6try ~tandard 96-well microtiter plate The multi-piece di~posable 25 plastic ~yctem is very tolerant of manufacturing tolerance errors and the differing thermal expansion rates over the wide temperature range ~ncounter-d during PCR thermal cycling Figures 2LA and 21B show alternative mbodiments of 30 most of the four pi-c- plastic ~ystem component~ in cross-~ection as assembled to hold a plurality of ~ample tubes in their sample wells with ~ufficient freedom of motion to account for differing rates of thermal ~xpancion Figure 45 shows all the parts of the di~po~able plastic microtiter 35 plate emulation system in an exploded view This figure illustrates how the parts fit together to form a microtiter - ~4 -plate with all the ~ample tubes loosely retained in an 8X12 microtit-r plate format 96 well array Figure 22 shows a plan view of a microtiter plate frame 342 according to the teachings of the invention which ie partially ~hown in 5 cross-section in Figures 21A and 21B. Figure 23 shows a bottom view plan view of the frame 342. Figure 24 is an end view of the frame 342 taken from view line 24-24 in Figure 22. Figure 25 iS an end view of the frame 342 taken from view line 25-25 in Figure 22. Figure 26 i~ ~ cro-~ ~ection 10 through the frame 3~2 at ~ection line 26-26 in Figure 22.
Figure 27 is a cross ~ectional view-through the frame 342 taken along section line 27-27 in Figure 22. Figure 28 iS
a side view of the frame 342 taXen along view line 28-28 in Figure 22 with a partial cut away to show in more detail the 15 location where a retainer to be described below clip6 to the frame 342.
Referring jointly to Figures 21A 21B and 22 through 28 the frame 342 iS comprised of a horizontal plastic plate 372 in which there are formed 96 holes ~paced on 9 20 millimeter centers in the standard microtiter plate format There are 8 row- labeled A through H and 12 columns labeled 1 through 12. Hole 374 at row D, colu~n 7 i~ typical of these holes In each hole in the frame 342 there i~ placed a conical ~ample tube ~uch as the sample tube 376 shown in 25 Figure 15 Each sample tube is ~maller in diameter than the hole in which it is placed by about 0 7 millimeters, ~o that there i~ ~ loose fit in the hole This i~ be~t seen in Figure~ 21A and 21B by obcerving the distanc- b tw-en the inside edge 378 of a typical hol- and th- ~id- wall 380 of 30 the sample tube plac-d th-rein ~ferenc- nuneral 382 in Figures 21A and 21B show6 the opposite ~dge of the hole which is also ~paced away from the outcide wall of the cylindrical portion of the sampl- tube 376.
Each ~ample tube has a ~houlder ~hown at 384 in Figures 35 15 21A and 21B. This shoulder is molded around the entire circumference of the cylindrical portion 288 of each sample tube The diameter of this ~houlder 384 i- large ~nough that it will not pass through the holes in the frame 342, yet not ~o large as to touch the ~houlders of the adjacent tubes in n-ighboring holes Once all the tubes are placed in their holes in the frame 342, a plastic retainer 386 (best seen in Figures 21A
and 2lB and Figure 45) is snapped into apertures in the frame 342 The purpo6e of this retainer is to keep all the tubes in place ~uch that th-y cannot fall out or b- ~nocked 10 out of the frame 342 while not interfering with th-ir looseness of fit in the frame 342 The retainer 386 is sized and fitted to the fram- 342 ~uch that ~ach ~ample tube has freedom to ~ove v-rtically up and down to ~ome extent before the ~houlder 384 of the tub- encounters either the 15 retainer 386 or the frame 342 Thus, the frame and retainer, when coupled, provide a microtit-r platé format for up to 96 ~ampl- tubes but provide sufficient horizontal and vertical freedom such that each tube is free to find its best fit at all temperatures under the influence of the 20 minimum threshold force F in Figure 15 A more cl-ar vi-w of the ~ample tube and shoulder may be had by referenc- to Figures 29 and 30 Figures 29 and 30 are an el-vation aectional view and a partial upper s-ction of t~e shoulder portion, respectively, of a typical sample 25 tube A plastic dome-~haped cap such as will be described in more detail below is in~erted into the sample tube shown in Figure 29 and forms a hermetic ~eal with the insid- wall 390 of the top at the ~ample tube A ridge 392 formed in the inside wall of the ~ample tube act- as a stop for the 30 dome-shaped cap to prevent further penetration Normally, the dome-shaped caps come in ~trips connected by web Figure 31 ~hows three caps in elevation view connected by a web 394 and terminated in a tab 396 The tab aids the user in removing an entire row of caps by a single pull 35 ~ormally, the web 394 rests on the top ~urface 398 of the sample tube and prcvents further penetration of the cap into the sample tube Each cap includes a ridge 400 which forms the hermetic seal between the cap and th- inside wall of the sample tube Figure 32 shows a top view of three caps in a typical strip of 12 connected caps For a more detailed understanding of the retainer, refer to Figures 33 through 37 Figure 33 is a top view of the plastic retainer Figure 34 i~ an elevation view of the retainer taken along view line 34-34' in Figure 33 Figure 35 is an cnd ~levation view of the retainer taken along view lo line 35-35' in Figure 33 Figure 36 i~ a ~ectional view taken along section line 36-36' in Figur- 33 Figure 3~ is a sectional vicw through the retainer taken along ~ection line 37-37' in Figure 33 Referring jointly to Figure- 33-37, the retainer 386 i~
15 compri~ed of a ~ingle horizontal plastic plane 402 surrounded by a vertical wall 404 Th- plane 402 has an 8 x 12 array of 96 hole~ formed therein divided into 24 groups of four hol-s per group These groups are cet off by ridges formed in the plane 402 such as ridges 406 and 408 Each 20 hole, of which hole 410 i~ typical, has a diameter D which is larger than the diameter D~ in Fig 29 and ~maller than the diameter D2 Thi- allows th- r-tainer to be clipp-d over the ~ample tubes after they have been placed in the frame 342 but prevents the ~ample tubes from falling out of the 25 frame ~inc- the ~houlder 384 ic too large to pass through the hol- 410 The retainer ~naps into the frame 342 by means of plastic tab6 414 ~hown in Figure~ 34 and 36 These plastic tabs ar- pu~hed through the ~lot~ ~16 and ~18 in the frame 30 as shown in Figur- 23 There ar- two pla~tic tab~ ~14, one on each long edge of the r-tainer These two plastic tabs are ~hown as 414A and 414B in Figure 33 The frame 342 of Figures 22-28, with up to 96 cample tubes placed therein and with the retainer 386 ~napped into 35 place, forms a single unit cuch as i~ chown in Figures 21A
and 2lB ~hich can be placed in the sample block 12 for PCR

processing After processing, all the tubes may be removed simultaneou~ly by liftinq the frame 342 out of th- cample bloc~ For convenience and storage, the frame 342 with 5 sample tubes and retainer in place can be in6erted into another plastic component called the ba6e The base has the outside dimensions and footprint of a ~tandard 96-well microtiter plat- and iB shown in Figure 38 t~rough 44 Figure 38 is a top plan view of th- ba~e 420, whil- Figure 10 39 is a bottom plan view of th- ba-- Figur- 40 i~ an elevation view of the base taken from view lin- 40-40' in Figure 38 Figure 41 i~ an end el-vation view taken from view line 41-41' in Figure 38 Figure ~2 is a ~-ctional view taken through the ba~e along ~ection line 42-42' in 15 Figure 38 Figure 43 i- a ~ectional vi-w through the base taken along ~ection line 43-43' in Figur- 38 Figure 44 is a ~ectional view taken along ~ection line 44-44' in Figure The base 420 includes a flat plane 422 of plastic in 20 which an 8 x 12 array of holes with ~loped ~dges i~ formed These hole6 hav- dim-nsions and ~pacing ~uch that when the frame 342 i~ ~-at-d in th- ba--, th- botto~s of th- ~ample tubes fit into the conical hole- in th- ba-e ~uch that the sample tube~ are held in the ~ame relationship to the frame 25 342 a~ the ~mple tube~ ar- h-ld wh-n the frame 342 is mounted on the ~ample block Hole 424 i~ typical of the 96 hole~ formed in the ba~e and i~ ~hown in Figur~ 38, ~4 and 43 Th- individual ~ampl- tube~ o~ loo~ely captur-d between the tray and r-tain-r, b come fir~ly r-ated and 30 immobile when the frame i~ in-ert-d in the ba~e The manner in which a typical cample tube 424 fit~ in th- ba~e i~ ~hown in Figure 44 In other words, when th- frame, ~ample tube~ and retainer are ~eated in the ba6e 420 the ~ntir- as6embly 35 becomes the exact functional eguival-nt of an indus~ry standard 96-well microtiter platc, and can be placed in virtually any automated pipetting or ~ampling sy6tem for 96-well industry ~tandard microtiter plates for further processing After the ~ample tubes hav- been fill-d with the 5 necessary reagents and DNA ~ample to be amplified, the sample tubes can be capped In an alternative embodiment of the cap strip shwon in Figures 31 and 32, an entire mat of 96 caps with a compliant web connecting th-m in an 8 X 12 array may be used This web, ahown at 394 in Figure 31 must 10 be sufficiently compliant 80 that the cap- do not rectrain the sample tubes from making th- s~all motion- th~-e sample tubes must ~ake to fit perf-ctly in th- conical wells of the sample block at all temperature6 Th- as-embly of tubes, caps framcs, retainer and base 15 is brought after filling the tub-s to the thermal cycler There, the fr~e, capped tu~es and r-tainer plate are removed from the base as a unit Thi- unit is then placed in the ~ample block 12 to mak~ the a--embly ~hown in Figure 21A or 21B with the tubes 1008ely h-ld in the conical wells 20 in the ~ample block As chown in Figure 21, the frame 342 is ceated on th- top ~urface 280 of the guardband ln the preferred embodiment, the ridge 366 axtend- down into the groove 78 of the guardband, but this is not essential Next, the heated cover is slid over the ~amples, and 25 the heated platen is ~crewed down as prev$ously described until it contacts the top edge 346 of th- frame 342 Within ~-conds after the heat-d plat-n 14 in Figur- 19 touches th- cap-, the caps begin to ~often and yi-ld under the downward pr-s-ur- from the l-ad acrew 312 in Figure 19 30 The user then continu-- to turn to knob 318 until the index marks 332 and 334 in Figure 20 line up which indicates that every sample tube ha- been tightly press-d into the ~ample block with at least the minimum threshold force F and all air gaps between the heated platen 14, the ~ample block and 35 the top edge 346 of the fra~e 342 have been tightly closed The sample tubes are now in a completely closed and _ 79 _ controlled environment, and precision cycling of temperature can begin At the end of the PCR protocol, the h-ated platen 14 is moved upward and away from tb- ~ampl- tubes, and the heated 5 cover 316 is ~lid out of the way to expose the frame 342 and sample tubes The frame, sample tubes and retainer are then removed and replaced into an empty base, and the caps can be removed As each cap or string of caps is pulled off, the retainer keeps the tube from coming out of the tray Ribs 10 formed in the base (not shown in Figures 38-44) contact the retainer tabs 414A and 414B shown in Figure 33 to keep the retainer snapped in place such that the force exerted on the tubes by removing the caps do-- not dislodge the retainer Obviously, the frame 342 may be u~ed Yith fewer than 96 tubes if desired Also, the retainer 386 can be removed if desired by unsnapping it A user who wishes to run only a f-w tub~s at a time and handle these tubes individually can plac- an ~mpty frame 342 20 without retainer on the sample block The user m~y then use the base a- a ~test tube rack~ and ~et up a small number of tubes therein The~e tubes can tb-n be filled manu~lly and capped with individual caps The user may then transfer the tubes individually $nto wells in tbe sample block, close the 25 heated cov-r and ~crew down th- h-at-d plat~n 14 until the m~rks line up PCR cycling Day then co~mence When the cycling is complete, the cover 316 i- r-mov-d and the sample tubes ar- individually plac-d in an available base Tbe retainer is not n-c~ ry in thi- type of u-ag-Referring to Figures 47A and 47~ (hereafter Figure 47), there is shown a block diagram for the ~l-ctronics of a preferred embodiment of a control ~y-t-m in ~ class of control ~ystems represent-d by CPU block 10 in Figure 1 The purpose of the control el-ctronic- of Figure 47 is, 35 inter alia, to r-ceive and ~tor- us~r input data defining the desired PCR protocol, read the various temperature sensors, calculate the sample temperature, compare the calculated sample temperature to the desired t~mperature as defined by the user defin-d PCR protocol, ~onitor th- power line voltage and control the film heater zones and the ramp 5 cooling valves to carry out the desired temperature profile of the user defined PCR protocol A microprocessor (hereafter CPU) 450 executes the control program described below and given in ~ ndi~ C in source code form In the preferred embodi~ent, the CPU 450 10 is an OKI CMOS 8085 The CPU drives an addre~ bu- 452 by which various ones of the other circuit ~lement~ in Figure 47 are addressed The CPU also drives a data bus ~54 by which dat~ i- transmitted to various of the other circuit element~ in Figure 47 The control program of Appendix C and ~ome system constant~ are stor-d in EPROM 456 User ~nt-r-d data and other ~yst-m constants and characteristic~ mea6ured during the install process (install program ex-cution described below) are stored in batt-ry back-d up RAM 458 A ~ystem 20 clock/calendar 460 ~upplies the CPU 450 with date and time information for purpose~ of r-cording a hi-tory of events during PCR run~ and the duration of power failures as described below in the description of the control software An address d-cod-r 462 r-ceives and d-code- ~ddresses 25 from th- addr--- bu- 452 and activates the appropriate chip ~el-ct line~ on a chip ~elect bu~ 464 The u~er enter~ PCR protocol data via a k-yboard 466 in re~pon~- to inforoation di~played by CPU on display 468 The two way comnunic~tion b tween the u~er and the CPU 450 30 is described in more detail below in the us-r interface section of the description of the control ~oftware A
keyboard interface circuit 470 converts u~er keystrokes to data which is read by the CPU via the data bu~ 454 Two program~abl- int-rval timerc 472 and 474 each 3S contain counters which are loaded with count- calculated by the CPU 450 to control the intervals during ~hich power is applied to the various film heater zones An interrupt controller 476 ~ends interrupt reque-ts to the CPU 450 every 200 milliseconde causing th- CPU 450 to run the PID task described b low in th- d--cription of the 5 control ~oftware This task r-ads the temperature sensors and calculates the heating or cooling power necessary to move the sample temperature from its current level to the level desired by the user for that point in time in the PCR
protocol being executed A UART 478 ~ervicc~ an RS232 int-rfac- circuit 480 ~uch that data stored in the RAM 480 may be output to a printer The control software maintain- a r-cord of each PCR run which is performed with respect to the actual temperatures which existed at various times during the run for pu~poses 15 of user validation that the PCR protocol actually executed corresponded to the PCR protocol desir-d by the u~er In addition user entered data defin$ng the specific times and temperatures desired during a particular PCR protocol is also stored All thi- data and other data as well ~ay be 20 read by the CPU 450 and ou~y~ to a printer coupled to the RS232 port via the UART 478 Th- RS232 interface also allows an ~xternal computer to tak- control of the address and data buse~ for purposes of testing A peripheral interface chip (her-after PIC) 482 serves 25 as a programmable ~et of 4 input/output r-gisters At power-up the CPU 450 ael-ct- the PIC 482 via the address decoAi~r 462 ~nd the chip ~ ct bua 464 The CPU then writes a data word to the PIC via data bu- 454 to ~o~am the PIC 482 regarding wh$ch r-gister~ are to b output ports 30 and which are to be input ports Sub~-qu-ntly the CPU 450 uses the output registers to ~tore data words written therein by the CPU via th- data bus 454 to control the internal logic ~tate of a ~Gy~ammable ~rray logic chip (PAL) 484 The PAL 484 is a ~tate machine ~hich has a plurality of input signals and ~ plurality of output signals PAL s in general contain an array of logic which has a number of different states Each state i5 d-fin-d by the array or vector of logic states at the input~ and ~ach ~tat- results in a different array or vector of logic ~tat-s on the 5 outputs The CPU 450, PIC 482, PAL 484 and ~everal other circuits to be defined below cooperate to generate different states of the various output ~ignals from the PAL 484 These different states and as60ciated output ~ign~ls are what control the operation of the ~l-ctronic~ ~hown in 10 Figure 47 as will be de~crib d b low A 12 bit analog-to-digital converter (A/D) 486 converts analog voltages on line- 488 and 490 to digital signals on data bus 454 These are read by the CPU by generating an address for the A/D converter such that a chip ~elect signal 15 on bus 464 coupled to the chip ~elact input of the A/D
converter goes active and activates the converter The analog signals on lines 488 and 490 ~re the output lines of two multipl-xer6 492 and 494 Multipl-xar 492 ha- four inputs ports, ~ach having two ~ignal lin-- Each of these 20 port- i- coupled to one of the four temperature ~-n-ors in the ~ystem The first port is coupled to the ~mple block temperature ~enaor Tha ~econd and third port- ar- coupled to the coolant and ambient temperature ~ensors, re~pectively and the fourth port is coupled to th- heated cover 25 temperatur- ~-n~cr A typical circuit for ~ach on- of these t-~p-rature ~ensors ls ~hown in Figure 48 A 20,000 ohm resi-tor 496 r-ceive- at ~ node 497 a r-gulated ~15 volt regulat-d power ~upply 498 in Figur- 47 vi~ a bus connection line w~ich is not ahown This +15 ~olt- D C signal reverse 30 biases a z-ner diode S00 The rever~e bias current and the voltage drop acroc- the zener diode ~r- functions of the temperature T~- voltage drop ~cross the ~iode is input to the rultiplexer 292 via lin-s 502 ~nd S04 Each t-mp-rature sensor hac a similar conn-ction to the multiplexer 292 Multiplexer 494 also hac 4 input port- but only t~ree are connected T~e first input port is coupl-d to a calibration volt~ge generator 506 This voltage generator outputs two precisely controlled voltage l-v-ls to the multiplexer inputs and i~ very thermally stable That is, the reference voltage output by voltage ~ource 506 drift~
5 very little if at all with temperature This voltage is read from time to time by the CPU 450 and compared to a stored constant which represents the level this reference voltage had at a known temperature as ~easur-d during ~xecution of the in-tall ~c~ de-crib-d below If the 10 reference voltage has drifted fro~ the l-vel ~easured and stored during the install proce-s, the CPU 450 knows that the other el-ctronic circuitry used for ~ensing the various temperatures and line voltage~ has also drifted and adjusts their outputs accordingly to maintain very accurate control 15 over the temperature measuring process The other input to the rultipl-x-r 494 i- coupled via line 510 to an RMS-to-DC converter circuit 512 This circuit has an input 514 coupled to a st-p-down transformer 516 and receives an A C voltage at input S14 which i~
20 proportional to the then existing line voltage at A C power input 518 The RMS-to-DC converter 512 rectifies the A C
voltage and averages it to develop a D C voltage on line 510 w~ich al~o i- proportional to th- A C input voltage on line 518 Four optically coupl-d triac driv-r- 530, 532, 534 and 536 r-ceive input control signals via control bus S38 from PAL logic ~84 Each of th- triac drivers 530, 532 and 534 controls power to one of the three film h-at-r zone- These heater zones ar- represented by blocks 2S4, 260/262 and 30 256t258 (the same reference numerals us-d in Figure 13) The triac driver 536 controls power to the h-~t-d cover, represented by block 544 via a thermal cut-out ~witch 546 ~he heater zone- of the film heat-r are prot-ct-d by a block thermal cutout switch 548 The purpose of the thermal 35 cutout switche is to prevent meltdown of the film heater/sample block on the heated cover in case o~ a failure ,. . .

leading to the triac drivers being left on for an unsafe interval. If such an event happens, the thermal cut-out switches detect an overly hot condition, and shut down the triacs via signals on lines 552 or 554.
The main heater zone of the film heater is rated at 360 watts while the manifold and edge heater zones are rated at 180 watts and 170 watts respectively. The triac drivers are Motorola MAC
15A10 15 amp triacs. Each heater zone is split into 2 electrically isolated sections each dissipating ~ the power. The 2 halves are connected in parallel for line voltages at 518 less than 150 volts RMS. For line voltages greater than this, the two halves are connected in series. These alternate connections are accomplished through a "personality" plug 550.
The AC power supply for the film heater zones is line 559, and the AC supply for the heated cover is via line 560.
A zero crossing detector 566 provides basic system timing by emitting a pulse on line 568 at each zero crossing of the AC
power on line 518. The zero crossing detector is a National LM
311N referenced to analog ground and has 25 mV of hysteresis.
The zero crossing detector takes its input from transformer 516 which outputs A.C. signal from 0 to 5.52 volts for an A.C. input signal of from 0 to 240 volts A.C.
A power transformer 570 supplies A.C. power to the pump 41 that pumps coolant through the ramp and bias cooling channels.
The refrigeration unit 40 also receives its A.C. power from the transformer 570 via another portion of the personality plug 550.
The transformer 550 alsQ supplies power to three regulated power supplies 572, 498 and 574 and one unregulated power supply 576.
For accuracy purposes in measuring the temperatures, the calibration voltage generator 506 uses a series of very precise, thin-film, ultralow temperature drift 20K ohm resistors (not shown in Figure 47). These same ultralow drift ., resistors are used to set the gain of an analog ~mplifier 578 which ~mplifies the output voltage from the ~elected temperature eensor prior to conversion to a digital value These resistors drift only 5 ppm/C~
All the temperature sensor~ are calibrated by placing them (separated from the 6tructures whose temperatures they measure) first in a ~table, 6tirred-oil, temperature controlled bath at 40~C and mea6uring the actual output voltage~ at the inputs to the multiplexer 492 The 10 temperature sensors are then placed in a bath at a temperature of 95~C and their output voltages are again measured at the ~ame point6 The output voltage of the calibration voltage generator 506 ic also measured at the input of the multiplexer 494 For ~ach t-mperature, the 15 digital output difference from the A/D converter 486 between each of the t-mperature 6ensor outputs and the digital output that results from the voltage generated by the calibration voltage generator 506 i6 measured The calibration constants for each temperature censor to 20 calibrate each for changes in temperature may then be calculated The sample block temperature cen60r is then subjected to a further calibration procedure This procedure involves driving the ~ample block to two different temperatures At 25 each temperature level, the actual temperature of the block in 16 different sample wells i6 mea6ured using 16 RTD
thermocouple probes accurate to within 0 02 C An average profile for the temperature of th- block i~ then generated and the ou~u~ of the A/D converter 464 i~ ~ea6ured with the 30 block temperature sensor in it~ place in the ~ample block Thi6 i~ done at both temperature level6 From the actual block temperature a~ measured by the RTD probes and the A/D
output for the block temperature ~ensor, a further calibration factor can be calculated The temperature 35 calibration factors so generated are ~tor-d in battery backed up RAM 458 Once these calibration factors are ~, ~

determined for the system, it is important that the system not drift appreciably from the electrical characteristics that existed at the time of calibration. It is important therefore that low drift circuits be selected and that ultralow drift resistors be used.

The manner in which the CPU 450 controls the sample block temperature can be best understood by reference to the section below describing the control program. However, to illustrate how the electronic circuitry of Figure 47 cooperates with the control software to carry out a PCR protocol consider the following.
The zero crossing detector 566 has two outputs in output bus 568. One of these outputs emits a negative going pulse for every positive going transition of the A.C. signal across the zero voltage reference. The other emits a negative pulse upon every negative-going transition of the A.C. signal across the zero reference voltage level. These two pulses, shown typically at 580 define one complete cycle or two half cycles. It is the pulse trains on bus 568 which define the 200 millisecond sample periods. For 60 cycle/sec A.C. as found in the U.S., 200 milliseconds contain 24 half cycles.
A typical sample period is shown in Figure 49. Each "tick"
mark in Figure 49 represents one half cycle. During each 200 msec sample period, the CPU 450 is calculating the amount of heating or cooling power needed to maintain the sample block temperature at a user defined setpoint or incubation temperature or to move the block temperature to a new temperature depending upon where in the PCR protocol time line the particular sample period lies. The amount of power needed in each film heater zone is converted into a number of half cycles each heater zone is to remain off during the next 200 msec sample period. Just before the end of the current sample period in which these calculations are ~ I ~

made, the CPU 450 addresses each of the 4 timers in the programmable interval ti~er (PIT) 472 To ~ach timer, the CPU writes data constituting a ~present" count representinq the nu~ber of half cycles the heater zone associated with 5 that timer is to remain off in the next ~ample period In Figure 49, this data is written to the timers during interval 590 just preceding the starting time 592 of the next sample period Assume that a rapid ra~p up to the denaturation temperature of 94~C is called for by the u--r 10 setpoint data for an interval which include6 the ~ample interval between times 592 and 594 Accordingly, the film heater6 will be on for mo~t of the period Ac6ume that the central zone heater is to be on for all but three of the half cycles during the ~ample period ~n thic ca6e, the CPU
15 450 write~ a three into the counter in PIT 472 ~ssociated with the central zone heater during interval 590 This write operation automatically causes the timer to issue a ~shut off" signal on the particular control line of bus 592 which controls the central zone heater This ~-hut off"
20 signal causes the PAL 484 to is~ue a ~hut off" signal on the particular one of the cignal lines in bu~ 538 as~ociated with the c-ntral zone The triac driver 530 then ~huts off at the next zero cros6ing, i e , at time 592 The PIT
r-ceives a pulse train of positive-going pul-es on line 594 25 from the PAL 484 These p~l-e~ are tr~nclations of the ze~o _~o~sing pul~es on 2-line bus 568 by PA~ 484 into positive going pulses at all zero cro~sing pul-e~ on 2-line bus 568 by PAL 484 into po~itiv- going pul ~t~ at all zero crossings on a ~ingle line, i - , line 594 The timer in 30 PIT 472 associated with the central film heatcr zone ~tarts counting down from its present count of 3 using the half cycle marking pulse6 on line 594 a~ its clock At the end of the third half cycle, thi6 timer reach-6 0 and causes its output ~ignal line on bus 592 to change stat-~ This 35 transition from the off to on ~tate is ~hown at 596 in Figure 49 This transition is communicated to PAL 484 and . .

1, .

c~uses it to change the 6tate of the appropriate output signal on bus 538 to switch the triac driver 530 on at the third zero-crossing Note that by ~witching the triacs on at the zero crossings as is done in the preferred S e~bodiment, switching off of ~ high current flowing through an inductor (the film heater conductor) is avoided This minimizes the generation of radio frequency interference or other noise Note that the technique of ~witching a portion ~f ~Çh half cycle to the film heater in accordanc~ with the 10 calculated amount of power needed will al-o work a~ an alternative embodiment, but i8 not preferr-d because of the noise generated by this technigue The other timers of PIT 472 and 474 work in a similar manner to manage the power applied to the other hcater zones 15 and to the heated cover in accordance with power calculated by the CPU
Ramp cooling is controlled by CPU 450 directly through the peripheral interface 482 When the h-ating/cooling power calculations performed during ~ach ~ample period 20 indicate that ramp cooling power is ~-E~-d, the CPU 450 addre~es the peripheral interface controller (PIC) 482 A
data word is then written into the appropriate register to drive output line 600 high This output line triggers a pair of monostable multivibrator6 602 and 604 and causes 25 ~ach to ~mit a cingle pulse, on line~ 606 and 608, re~pectively These pulse6 ~ach have peak current~ ~ust under 1 ampere and a pul6e duration of approximately 100 milli6econd6 The purpose of these pul~ to drive the 601enoid valve coils that control flow through the ramp 30 cooling channels very hard to turn on ramp cooling flow guickly The pulse on line 606 causes a driver 610 to ground a line 612 coupl-d to one side of the ~olenoid coil 614 of one of the 601enoid operated valve~ The other terminal of the coil 614 i~ coupled to a power ~upply ~rail"
35 616 at +24 volts DC from power ~upply 576 The one chot 602 controls the ramp cooling solenoid operted valve for flow in CA 022660l0 l999-04-Ol one direction, and the one shot 604 controls the solenoid operated valve for flow in the opposite direction.
SimultaneouSly, the activation of the RCOOL signal on line 600 causes a driver 618 to be activated. This driver grounds the line 612 through a current limiting resistor 620. The value of this current limiting resistor is such that the current flowing through line 622 iS at least equal to the hold current necessary to keep the solenoid valve 614 open. Solenoid coils have transient characteristics that require large currents to turn on a solenoid operated valve but substantially less current to keep the valve open. When the 100 msec pulse on line 606 subsides, the driver 612 ceases directly grounding the line 612 leaving only the ground connection through the resistor 620 and driver 618 for holding current.
The solenoid valve 614 controls the flow of ramp cooling coolant through the sample block in only ~ the ramp cooling tubes, i.e., the tubes carrying the coolant in one direction through the sample block. Another solenoid operated valve 624 controls the coolant flow of coolant through the sample block in the opposite direction. This valve 624 iS driven in exactly the same way as solenoid operated valve 614 by drivers 626 and 628, one shot 604 and line 608.
The need for ramp cooling is evaluated once every sample period. When the PID task of the control software determines from measuring the block temperature and comparing it to the desired block temperature that ramp cooling is no longer needed, the RCOOL signal on line 600 iS deactivated. This is done by the CPU 450 by addressing the PIC 482 and writing data to it which reverses the state of the appropriate bit in the register in PIC
482 which is coupled to line 600.

,, .

The PIT 474 also has two other timers therein which time a Hz interrupt and a heating LED which gives a visible indication when the sample block is hot and unsafe to touch.
The system also includes a beeper one shot 630 and a beeper 632 to warn the user when an incorrect keystroke has been made.
The programmable interrupt controller 476 is used to detect 7 interrupts; Level 1 - test; Level 2-20 Hz; Level 3 - Transmit Ready; Level 4 - Receive ready; Level 5 - Keyboard interrupt;
Level 6 - Main heater turn on; and, Level 7 - A.C. line zero cross.
The peripheral interface controller 482 has four outputs (not shown) for controlling the multiplexers 492 and 494. These signals MUX1 EN and MUX2 EN enable one or the other of the two multiplexers 492 and 494 while the signals MUX 0 and MUX 1 control which channel is selected for input to the amplifier 578.
these signals are managed so that only one channel from the two multiplexers can be selected at any one time.
An RLTRIG* signal resets a timeout one shot 632 for the heaters which disables the heaters via activation of the signal TIMEOUT EN* to the PAL 484 if the CPU crashes. That is, the one shot 632 has a predetermined interval which it will wait after each trigger before it activates the signal TIMEOUT EN* which disables all the heater zones. The CPU 450 executes a routine periodically which addresses the PIC 482 and writes data to the appropriate register to cause activation of a signal on line 634 to trigger the one shot 632. If the CPU 450 "crashes" for any reason and does not execute this routine, the timeout one-shot 632 disables all the heater zones.
The PIC 482 also has outputs COVHTR EN* and BLKHTREN* (not shown) for enabling the heated cover and the sample block heater.
Both of these signals are active low and are controlled by the CPU 450. They are output to the PAL 484 via bus 636.
The PIC 482 also outputs the signals BEEP and BEEPCLR*
on bus 640 to control the beeper one shot 630.
The PIC 482 also outputs a signal MEM1 (not ~hown) which is used to switch pages between the high address section of EPROM 456 and the low address section of battery RAM 458. Two other signals PAGE SEL 0 and PAGE SEL 1 (not shown) are output to select between four 16K pages in EPROM
10 456.
The four temperature sensors are National LM 135 zener diode type ~ensors with a zener voltage/temperature dependence of 10 mV/~K. The zener diodes are driven from the regulated power supply 498 through the 20X resistor 496.
15 The current through the zeners varies from approximately 560 ~A to 615 ~A over the 0~C to 100~C operating range. The zener self heating varies from 1.68 mW to 2.10 mW over the same range.
The multiplexers 492 and 494 are DG409 analog switches.
20 The voltages on lines 488 and 490 are amplified by an AD625KN instrumentation amplifier with a transfer function of Von- 3~VI~ - 7.5. The A/D converter 486 is an AD7672 with an input range from 0-5 voltc. With the zener temperature sensor output from 2.73 to 3.73 volts over the 0~C to 100~C
25 range, the output of the amplifier 578 will be 0.69 volts to 3.69 volts, which is comfortably within the A/D input range.
The key to highly accurate system performance are good accuracy and low drift with changes in ambient temperature.
~oth of these goals are achieved by using a precision 30 voltage reference source, i.e., calibrAtion voltage generator 506, ~nd continuously monitoring its output through the same chain of electronics as are used to monitor the outputs of the temperature sensors and the AC line voltage on line 510.
The calibration voltage generator 506 outputs two precision voltages on lines 650 and 652. One voltage is ~ . , 3.75 volts and the other is 3.125 volts. These voltages are obtained by dividing down a regulated supply voltage using a string of ultralow drift, integrated, thin film resistors with a 0.05~ match between resistors and a 5 ppm/degree C temperature drift coefficient between resistors. The calibration voltage generator also generates -5 volts for the A/D converter reference voltage and -7.5 volts for the instrumentation amplifier offset.
These two voltages are communicated to the A/D 486 and the amplifier 578 by lines which are not shown. These two negative voltages are generated using the same thin film resistor network and OP 27 GZ op-amps (not shown). The gain setting resistors for the operational amplifier 578 are also the ultralow drift, thin-film, integrated, matched resistors.
The control firmware, control electronics and the block design are designed such that well-to-well and instrument-to-instrument transportability of PCR protocols is possible.
High throughput laboratories benefit from instruments which are easy to use for a wide spectrum of lab personnel and which require a minimal amount of training. The software for the invention was developed to handle complex PCR thermocycling protocols while remaining easy to program. In addition, it is provided -with safeguards to assure the integrity of samples during power interruptions, and can document the detailed events of each run in safe memory.
After completing power-up self-checks shown in Figures 53 and 54, to assure the operator that the system is operating properly, the user interface of the invention offers a simple, top-level menu, inviting the user to run, create or edit a file, or to access a utility function. No programming skills are required, since pre-existing default files can be quickly edited with customized times and temperatures, then stored in memory for later use. A file protection scheme prevents unauthorized changes to any user's programs. A file normally consists of a set of instructions to hold a desired temperature or to thermocycle Complex programs are created by linking files together to form a mothod A commonly used file, such as a 4~C incubation following a thermocycle, can be stored and then incorporated into methods created by 5 other users A new type of file, the AUTO file is a PCR
cycling program which _llows the user to specify which of several types of changes to control parameters will occur each cycle time incrementing (auto segment extension, for yield enhancement), time decrementing, or temperature 10 incrementing or decrementing For the highest ~r,~ee of control precision and most reliable methods transferability, temperatures _re setable to O l C, and times are progr_mmed to the nearest second The invention has the ability to program a scheduled PAUSE at one or more s-tpoints during a 15 run for reagent additions or for removal of tubes at specific cycles The system of the invention has the ability to store a 500 record history file for each run This feature allows the user to review the individual cteps in each cycle and to 20 flag any special status or error mes-ages relating to irre-gularities With the optional printer, the invention provides hardcopy documentation of file and method parameters, run-time time/temperature data with _ time/date stamp, configuration parameters, and sorted file 25 directories In order to assure ~t~od~cible thermocycling, the computed ample temperature is displayed during the ramp and hold segments of each cycle A temperature one degr-e lower than the set temper_ture is normally used to trigger the 30 ramp-time and hold-time clocks, but this can be altered by the user Provided the proper time constant for the type of tube _nd volume is used, the sample will always approach the desired sample temperature with the same accuracy, regardless of whether long or short sample inc~)hation times 35 have been programmed Users can program slow ramps for the specialized annealing requirements of degenerate primer . .

pools, or very short (1-5 sec) high-temperature denaturation periods for very GC rich targets. Intelligent defaults are preprogrammed for 2- and 3-temperature PCR cycles.
Diagnostic tests can be accessed by any users to check the heating and cooling system status, since the software gives Pass/Fail reports. In addition, a system performance program performs a comprehensive subsystem evaluation and generates a summary status report.
The control firmware is comprised of several sections which are listed below:
- Diagnostics - Calibration - Install - Real time operating system - Nine prioritized tasks that manage the system - Start-up sequence - User interface The various sections of the firmware will be described with either textual description, pseudocode or both.

Features of the firmware are:

1. A Control system that manages the average sample block temperature to within +/- 0.1~C as well as maintaining the temperature non-uniformity as between wells in the sample block to within +/- 0.5~C.

2. A temperature control system that measures and compensates for line voltage fluctuations and electronic temperature drift.

3. Extensive power up diagnostics that determine if system components are working.

4. Comprehensive diagnostics in the install program which qualify the heating and cooling systems to insure they are working properly.

5. A logical and organized user interface, employing a menu driven system that allows instrument operation with minimal depenaency on the operators manual.

6. The ability to link up to 17 PCR protocols and store them as a method.

7. The ability to store up to 150 PCR protocols and methods in the user interface.

8. A history file that records up to 500 events of the previous run as part of the seguence task.

9. The ability to define the reaction volume and tube size type at the start of a run for maximum temperature accuracy and control as part of the user interface and which modifies tau (the tube time constant) in the PID task.

10. Upon recovery from a power failure, the system drives the sample block to 4~C to save any samples that may be loaded in the sample compartment. The analyzer also reports the duration of the power failure as part of the seguence task.

11. The ability to print history file contents, "run time" parameters and stored PCR protocol parameters as part of the print task.

-95a-, 12. The ability to configure to which the apparatus will return during any idle state.

13. The ability to check that the set point temperature is reached with a reasonable amount of time.

14. The ability to control the instrument remotely via an RS232 port.

~ , .. .

There are several levels of diagnostics which are described below A series of power-up tests are automatically performed each time the instrument is turned on They evaluate 5 critical areas of the hardware without user intervention Any test that detects a component failure will be run again If the test fails twice, an error message is displayed and the keyboard is electronically locked to prevent the user from continuing The following areas are tested Programmable Peripheral Interface device Battery RAM device Battery RAM checksum EPROM devices Programmable Interface Timer devices Clock / Calendar device Programmable Interrupt Controller device Analog to Digital ~ection Temperature sensor6 Verify proper configuration plug A Series of ~ervice only diagno~tics are available to final testers at the manufacturer'~ location or to field service engineers through a ~hidden" key~troke ~equence (i e unknown to the customer) MAny of the te~t6 are the 25 same as the ones in the ~tart up diagnostic~ with the exception that they can be continually ~xecuted up to 99 times The following areas are tested Programmable Peripheral Interface device Battery RAM device Battery RAM checksum EPROM devices Programmable lnterface Timer devices Clock / Calendar device Programmable Interrupt Controller device Analog to Digital section RS-232 ~ection Display ~ection Keyboard Beeper Ramp Cooling Valves Check for EPROM mi~match Firmware version level Battery RAM Checksum and Initialization Autostart Program Flag Clear Calibration ~lag Heated Cover heater and control circuitry Edge heater ~nd control circuitry Manifold heater and control circuitry Central heater and control circuitry Sample block thermal cutoff test Heated cover thermal cutoff test User diagnostics are also available to allow the user to perform a guick cool and heat ramp verification test and an extensive confirmation of the heating and cooling system.
These diagnostics also allow the user to view the history file, which i~ a seguential record of event~ that occurred 25 in the previous run. The records contain time, temperature, setpoint number, cycle number, program number and 6tatus messages.
Remote Diagnostics are available to allow control of the system from an external computer via the RS-232 port.
30 Control is limited to the service diagnostic6 and instrument calibration only.
Calibration to determine various parameters such as heater resi6tance, etc. is performed. Access to the calibration screen is limited by a ~hidden" key ~equence 35 (i.e. unknown to the customer). The following parameters are calibrated:
The configuration plug is a module that rewires the chiller unit, sample block heaters, coolant pump and power supplies for the proper voltage and frequency (lOOV/50~z, 5 100/60Hz, 120/60Hz, 220/50Hz or 230/50Hz). The user enters the type of configuration plug installed. The firmware uses this information to compute the equivalent resistance of the sample block heaters. Upon power-up, the system verifies that the configuration plug selected is consistent with the 10 current line voltage and freguency.
The heater resistance must be determined in the calibration process so that precise calculations of heater power delivered can be made. The user enters the actual resistances of the six sample block heaters (two main 15 heaters, two manifold heaters and two edge heaters). The configuration plug physically wires the heater in series for 220-230 VAC and in parallel for 100-120 VAC operation. The firmware computes the equivalent resistance of each of the three heaters by the following formula:

(7) For 100-120 VAC: R~ s (Rl * R2) / Rl + R2 (8) For 220-230 VAC: Rq - Rl + R2 The equivalent resistance is used to deliver a precise amount of heating power to the sample block (Power - Voltage2 x Resistance).
The calibration of the A/D circuit is necessary so that temperatures can be precisely measured. This is performed by measuring two test point voltages (TP6 and TP7 on the CPU
board) and entering the measured voltages. The output of the A/D at each voltage forms the basis of a two point 30 calibration curve. These voltages are derived from a 5 volt precision source and are accurate and temperature independent. At the start of each run, these voltages are read by the system to measure electronic drift due to .. .

_ 99 _ temperature because any changes in A/D output i8 due to temperature dependencies in the analog chain (multiplexer, analog amplifier and A/D converter).
Calibration of the four temperature ~ensors (sample 5 block, ambient, coolant and heated cover) is performed for accurate temperature measurements. Prior to installation into an instrument, tbe ambient, coolant, and heated cover temperature sensors are placed in a water bath where their output is recorded (XX.X-C at YYYY mV). These values are 10 then entered into the ~ystem. Since temperature accuracy in these areas is not critical, a one point calibration curve is used.
The sample block sensor is calibrated in the instrument. An array of 15 accurate temperature probes is 15 strategically placed in the sample block in the prçferred embodiment. The output of the temperature probes is collected and averaged by a computer. The firmware commands the block to go to 40-C. After a brief stabilizing period the user enters the average block temperature a read by the 20 15 probes. This procedure is repeated at 95~C, forming a two point calibration curve.
Calibration of the AC to DC line voltage sampling circuit is performed by entering into the ~ystem the output of the AC to DC circuit for two given AC input voltages, 25 forming a two point calibration curve. The output of the circuit is not linear over the reguired range (90 - 260 VAC) and therefore requires two points at ~ach end (100 and 120, 220 and 240 VAC), but only uses one set based on the current input voltage.
An accurate measure of AC voltage is necessary to deliver a precise amount of power to the sample block tPower = Voltage2 x Resistance).
The Install program is a diagnostic tool that perfor~s an extensive test of the cooling and heating systems.
35 Install measures or calculates control cooling conductance, ramp cooling conductance at 10~C and 18~C, cooling power a~

10~C and 20~C, ~ample block thermal and coolant capacity and sample block ~ensor lag. The purpo~e of install is three fold:

1. To uncover marginal or faulty components.

2. To use some of the measured values as ~ystem constants ctored in battery backed up RAM to optimize the control ~ystem for a given instrument.

3. To mea6ure heating and cooling sy6tem degradation over time Inetall is executed once before the ~y~tem i6 shipped and 6hould also be run before uce or whenever a major component i6 replaced. The Install p~G~am may also be run by the u6er under the user diagnostics.
The heater ping te6t verifies that the heaters are properly configured for the current line voltage (i.e. in parallel for 90-132 VAC and in serie6 for 208-264 VAC). The firmware ~upplies a burst of power to the sample block and then monitors the ri~e in temperature over a 10 6econd time 20 period. If the temperature ri6e i6 outside a specified ramp rate window, then the heaters are incorrectly wired for the current line voltage and the install proce~s i~ terminated.
The control cooling conductance tests m-asures the thermal conductance Rcc across the sample block to the 25 control cooling passages. Thi~ test is performed by first driving the sample block temperature to 60-C (ramp valves are closed), then integrating the heater powcr required to maintain the block at 60~C over a 30 second time period.
The integrated power is divided by the ~um of the difference 30 between the block and coolant temperature over the interval.

(9) Rcc ~ r Heater Power 60'C / ~ Block - Coolant Temp Typical values are 1.40 to 1.55 Watts/~C. A low Kcc may indicate a clogged liner(s). A high Kcc may be due to a ra~p 5 valve that is not completely closed, leakage of the coolant to the outside diameter of the liner, or a liner that has shifted.
The block thermal capacity (Blk Cp) test measures the thermal capacity of the sample block-by first controlling 10 the block at 35~C then applying the maximum power to the heaters for 20 seconds. The block thermal capacity is equal to the integrated power divided by the difference in block temperature. To increase accuracy, the effect of bias - cooling power is subtracted from the integrated power.

(10) Blk Cp ~ ramp time ~ (heater - control cool pwr) / delta temp.
where:

ramp time - 20 seconds heater power ~ S00 watts control cool e (~ block - coolant temp) Rcc delta temp - TBlockt.20 ~ TBlockt.0 The typical value of Block Cp ic 540 watt-seconds/~C +
30. Assuming a normal Rcc value, an increase in block thermal 25 capacity is due to an increase in thermal loads, such as moisture in the foam backing, loss of insulation around the sample block, or a decrease in heater power such as a failure of one of the six heater zones or a failure of the electronic circuitry that drives the heater zones, or an 30 incorrect or an incorrectly wired voltage configuration module.

A chiller test measures the system cooling output in watts at 10~C and 18~C. The system cooling power, or chiller output, at a given temperature i6 ~qual to the summation of thermal loads at that temperature. The main S components are: 1. heating power required to maintain the block at a given temperature, 2. power dissipated by the pump used to circulate the coolant around the sy6tem, and 3.
losses in the coolant lines to the ambient. ~he chiller power parameter is measured by controlling th- coolant 10 temperature at either 10~C or 18-C and ~ntegrating the power applied to the sample block to maintain a constant coolant temperature, over a 32 second interval. The difference between the block and coolant temperature is also integrated to compute losses to ambient temperature.

(11) Chiller power - ~ Heating power + Pump power I (Kamb * ~ (blk-cool temp)) where:
heating power ~ Sum of heating power required to maintain coolant at 10~C
or 18~C over time 32 seconds.
Pump Power - Circulating pump, 12 watts Kamb - Conductance to ambient, 20 watts/-C
blk-cool temp ~ Sum of difference in block and coolant temp over time 32 ceconds The typical value for chiller power is 230 watts + 40 at 10~C and 370 watts + 30 at 18~C. Low chiller power may be due to an obstruction in the fan path, a defective fan, 30 or a marginal or faulty chiller unit. It may also be due to a miswired voltage configuration plug.
A ramp cooling conductance (Kc) test measures the thermal conductance at 10~C and 18~C across the sample block ..

to the ramp and control cooling passages This test is performed by first controlling the coolant temperature at 10~C or 18~C, then integrating, over a 30 6econd time interval, the heating power applied to maintain the coolant 5 at the given temperature divided by the difference of block and coolant temperature over the time interval (12) Rc - s Heating power / ~ (block - coolant temperature) Typical values for Kc are 28 watts/~C ~ 3 at 10~C and 31 10 watts/~C
+ 3 at 18~C A low Kc may be due to a closed or obstructed ramp valve, kinked coolant tubing, weak pump or a hard water/Prestone~ mixture A 6ensor lag test measures the block ~ensor lag by 15 first controlling the block temperature to 35~C and then applying 500 watts of heater power for 2 seconds and measuring the time required for the block to rise 1~C
Typical values are 13 to 16 units, where each unit is equal to 200 ms A slow or long sensor lag can be due to a poor 20 interface between the ~ensor and the block, ~uch as lack of thermal grease, a poorly machined sensor cavity or a faulty ~ensor The remaining-install tests are currently executed by the install program but have limited diagnostic pu~Dses due 25 to the fact that they are calculated values or are a function of so many variables that their results do not determine the cource of a problem accurately The install program calculates the ~lope of the ramp cooling conductance (Sc) between 18 C ~nd 10~C It i~ a 30 measure of the linearity of the conductance curve It is also used to approximate the ramp cooling conductance at 0~C ~ypical values are 0 40 + 0 2 The ~pread in values attest to the fact that it is just an approximation (13) Sc ~ (Kc 18~ - Kc 10~) / (18~C - 10~C) The install program also calculates the cooling conductance Kco~ Kco is an approximation of the cooling conductance at 0~C. The value is extrapolated from the 5 actual conductance at 10~C. Typical values are 23 watts/oc + 5. The formula used is:

(14) Kco - Kc 10 - (Sc ~ 10~C) The install program also calcul~tes coolant capacity (Cool Cp) which is an approximation of thermal capacity of 10 the entire coolant stream (coolant, plu~bing lines, heat exchanger, and valves). The cooling capacity is egual to components that pump heat into the coolant minus the components that remove heat from the coolant. The mechanics used to measure and calculate these components are complex 15 and are described in detail in the source code description section. In this measurement, the coolant i6 allowed to stabilize at 10~C. Maximu~ heater power is applied to the sample block for a period of 128 ~econds.

(15) Cool Cp - Heat Sources - Coolant sources 20 (16) Cool Cp - Neater Power + Pump Power + Kamb * (~Tamb -~Tcool) - Block Cp ~ (Tblockt,0 - Tblockt,12~) - Average Chiller Power between Tcoolt,O ~nd Tcoolp~2~

Characters enclosed in { } indicate the variable names used in the source code.

He~ter-Pin~ Test Pseudocode:
The heater ping test verifies that the heaters are properly wired for the current line voltage.

. .

Get the 6ample block and coolant to a known and 6table point.

Turn ON the ramp cooling valves Wait for the block and coolant to go below 5~C
Turn OFF ramp cooling valves Measure the cooling effect of control cooling by measuring the block temperature drop over a 10 second time interval. Wait 10 seconds for stabilization before taking any measurements.

Wait 10 seconds templ ~ block temperature Wait 10 6econds temp2 ~ block temperature {tempa} = temp2 - templ Examine the variable {linevolt~} which contains the actual measured line voltage. Pulse the heater with 75 watt6 for a line voltage greater then 190V or with 300 watts if it less than 140V.

if ({linevolts} ~ 190 Volts) then deliver 75 watts to heater else deliver 300 watts to heater Measure the temperature rise over a 10 ~econd time period. The result is the average heat rate in 0.01 ~/second.

templ ~ block temperature Wait 10 ~econds temp2 ~ block temperature {tempb} ~ temp2 - templ ., ~

Subtract the average heat rate {tempb} from the control cooling effect to calculate true heating rate (17) heat rate - {tempb} - {tempa}

Evaluate the heat rate For 220V-230V, the heat rate should be less than 0 30 ~/second For lOOV-120V the heat rate should be greater than 0 30 /second if (linevoltage - 220V and heat rate ~ 0 30 ~/6econd) then Error -> Heaters wired for 120 Lock up keyboard if (linevoltage - 120V and heat rate < 0 30 ~/second) then Error -~ Heaters wired for 220 Lock up keyboard 15 KCC Test Pseudocode This test measures the control cooling conductance also known as Kcc Kcc i~ mea~ured at a block temperature of 60~C

Drive block to 60~C
Maintain block temperature at 60~C for 300 ceconds lntegrate the power being applied to the 6ample block heaterc over a 30 eecond time period Measure and integrate the power reguired to maintain the block temperature with control cooling bias {dt 6um) - 0 (delta temperature ~um) {main pwr cum} ~ 0 (main heater power eum) {aux pwr sum} ~ 0 (auxiliary heater power eum) for (count ~ 1 to 30) {dt_sum} = {dt_sum} + (block temperature - coolant temperature) wait 1 sec Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the PID control task and is therefore summed every 200ms.

{main_pwr_sum} = {main_pwr_sum} + {actual_power}
{aux_pwr_sum) + {aux_pwr_sum} + {auxl_actual} +
{aux2_actual}
}

Compute the conductance by dividing the power sum by the temperature sum. Note that the units are 10 mW/~C.

(18) KCC = ({main_pwr_sum} + {aux_pwr_sum}) / {dt_sum}

BLOCK CP Test Pseudocode:
This test measures the sample block thermal capacity.

Drive the block to 35~C
Control block temperature at 35~C for 5 seconds and record initial temperature.

initial_temp = block temperature Deliver maximum power to heaters for 20 seconds while summing the difference in block to coolant temperature as well as heater power.

Deliver 500 watts {dt_sum} = o for (count ~ 1 to 20 6econds) {

{dt sum~ ~ ~dt sum} 1 (block temperature - coolant temperature) wait 1 second }

(19) delta temp ~ block temperature - initial temp Compute the joules in cooling power due to control cooling which occurs during ramp.

10 (20) cool joule ~ Control cooling conductance (Xcc) ~dt sum}

Compute the total joules applied to the block from the main heater and control cooling. Divide by temp change over the interval to compute thermal capacity.

15 (21) Block CP ~ ramptime ~ (heater power - cool joule) / delta temp -where: ramptime ~ 20 ~econds heater power - 500 WAtts COOL PWR 10:
20 This test measures the chiller power at 10~C.

Control the coolant temperature at 10-C and stabilize for 120 secs.

count ~ 120 do while (count !~ o) {
if (coolant temperature ~ 10 + 0.5-C) then count ~ count - 1 else count - 120 wait 1 second }

At this point, the coolant has been at 10~C for 120 seconds and has stabilized. Integrate, over 32 ~econds, the power being applied to maintain a coolant temperature of 10~C.

{cool_init} ~ coolant temperature {main_pwr sum} ~ o -{aux_pwr_sum} ~ 0 ~delta temp sum} - 0 for (count ~ l to 32) {
Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the control task.

{main pwr sum} - {main pwr ~um} + actual power {aux_pwr sum} ~ {aux pwr_sum} + auxl_actual +
aux2 actual delta temp sum - delta temp sum + (ambient temp -coolant temp) wait 1 second }
Compute the number of joules of energy added to the coolant mass during the integration interval. ~(coolant temp - cool init) n is the change in coolant temp during the integration interval. 550 is the Cp of the coolant in joules, t~us the product is in joules. It represents the extra heat added to the coolant which made it drift from 6etpoint during the integration interval This error is 6ubtracted below from the total heat applied before calculating the cooling power (22) cool init = (coolant temp - cool init) ~ 550J

Add the main power ~um to the aux heater ~um to get joules dissipated in 32 ~econds Divide by 32 to get the average joules/sec (23) {main pwr sum} ~ ({main pwr ~um}+{aux pwr sum} -cool init) / 32 Compute the chiller power at 10~C by ~umming all the chiller power components (24) PowerlO,c = main power 6um + PUMP PWR + (K AM3 delta temp 6um) where {main pwr sum~ ~ 6ummation of heater power over interval PUMP PWR ' 12 Watt6, pump that circulates coolant delta temp 6um ~ ~ummation of ~mb - coolant over - interval X AMB - 20 Watts/K, thermal conductance from cooling to ambient KC 10 Test Pseudocode This test measures the ramp cooling conductance at 10~C

Control the coolant temperature at lO C ~ O S and allow it to stabilize for 10 6econds At this point, the coolant is at 6etpoint and i6 being controlled. Integrate, over a 30 second time interval, the power being applied to the heaters to maintain the coolant at 10~C. Sum the difference between the block and coolant temperatures.

{main pwr sum} ~ 0 {aux pwr sum} - 0 {dt sum} - 0 for (count - 1 to 30) {

Accumulate the power applied to the main and auxiliary heaters. The actual code resides in the PID control task.

{main_pwr_sum} ~ {main_pwr_cum} + actual power {aux pwr sum} ~ {aux pwr_sum} + auxl_actual +
aux2 actual {dt sum} ~ {dt sum} + (block temperature - coolant temp) wait 1 second }

Compute the energy in joules delivered to the block over the summation period. Units are in 0.1 watts.

(25) {main_pwr_sum} ~ {main_pwr_sum} + {aux_pwr sum}

Divide the power sum by block - coolant temperature sum to get ramp cooling conductance in 100 mW/K.

25 (26) Kc 10 - {main_pwr sum} / ~dt sum}

COOL PWR 18 Test Pseudocode:

,.

This test measures the chiller power at 18~C .

Get the sample block and coolant to a known and ctable point. Control the coolant temperature at 18~C and stabilize for 128 secs.

count - 128 do while (count !- 0) if (coolant temperature ~ 18~C + 0.5) then count ~ count - 1 else count - 120 wait 1 cecond }

At this point the cool~nt has been at 18~C for 120 seconds and has 6tabilized. Integrate, over 32 seconds, the power being applied to maintain a coolant temperature of 18~C.

{cool init} - coolant temperature {main pwr ~um} - 0 ~aux_pwr cum} - 0 {delta temp 6um} - 0 for (count - 1 to 32) {

Accumulate the power appliod to the main and auxiliary heaters. ~he actual code resides in the control task.

{main pwr ~um} e {main pwr_~um} + actual power {aux_pwr_sum} - {aux pwr ~um} + auxl_actual +
aux2 actual delta temp ~um - delta_temp cum + (ambient temp -coolant temp) wait 1 second }

Compute the number of joules of energy added to the coolant mass during the integration interval. ~(coolant temp - cool init) n i~ the ch~nge in coolant temp during the integration interval. 550 i~ the Cp of the coolant in joules, thus the product i6 in ioule6. It represents the extra heat added to the cool~nt which made it drift setpoint during the integration interval. This error is subtracted below from the total heat ~pplied before calculating the cooling power.

(27) cool_init ~ (coolant temp - cool_init) ~ 550 Add ~ain power cum to aux heater 6um to get joules dissipated in 32 seconds. Divide by 32 to get the average joules/sec.

(28) {main pwr sum} - ({main_pwr_sum~+{aux_pwr cum}
cool init) / 32 Compute the chiller power at 18-C by summing ~11 the chiller power components.

(29) Powerl~.c - main power cum + PUMP PWR + (K AMB
delta temp cum) where:
{main pwr ~um} ~ summ~tion of heater power over interval PUMP PWR - 12 Watt~, pump that circulates coolant delta temp sum - summation of amb - coolant over interval K AMB ~ 20 Watt6/K, Thermal conductance from cooling to ambient.

5 KC 18 Test Pseudocode:
This test measure6 the ramp cooling conductance at 18~C.

Control the coolant temperature at 18~C I 0.5 and allow it to ~tabilize for 10 6econds.

At this point, the coolant i5 at ~etpoint and being controlled. Integrate, ov-r a 30 second time interval, the power being applied to the heaters to maintain the coolant at 18~C. Sum the difference between the block and coolant temperature.

{main pwr sum} - 0 {aux pwr sum} ~ 0 {dt 6um} ~ o for (count - 1 to 30) {

Accumulate the power applied to the main and auxiliary heater~. The actual code re~ides in the control task.

{main pwr_~um} - {main_pwr_~um} + actual_power {aux_pwr_sum} - {aux pwr_~um} + auxl actual +
aux2 actual {dt sum} - ~dt ~um} + (block temperature - coolant temp) wait 1 second }

Compute the energy in joules delivered to the block over the ~ummation period Units are in 0 1 watts (30) {main pwr ~um} - {~ain pwr ~um} I {aux_pwr sum}

Divide power ~um by block - coolant temperature sum to S get ramp cooling conductance in 100 mW/K

(31) Kc 18 - {main pwr 6um} / {dt sum}

SENT~G Test Pseudocode This test measures the sample block ~en~or lag Drive the block to 35~C Hold within ~ 0 2 C for 20 10 seconds then record temperature of block {tempa} ~ block temperature Deliver 500 watts of power to sample block Apply 500 watt~ of power for the next 2 ~-conds and count the amount of iterations through the loop for the block temperature to increase l C Each loop iteration execute~ every 200 ms, therefore actual ~ensor lag is egual to count ~ 200 ms ~ec~ - 0 count - 0 do while (TRUE) {

if (secs ~ 2 seconds) then ~hut heaters off if (block temperature - tempa > l O C) then exit while loop count ~ count + 1 }

end do while sensor lag - count 5 Coolant CP Test Pseudocode:
Thi- t-st compute~ the coolant capacity o~ the ~ntire system.
Stabilize the coolant temperature at 10~C + 0.5.

Send message to the PID control task to ramp the coolant temperature from it~ current value (a~out 10~C) to 18-C.

Wait for the coolant to cross 12~C so that the coolant CP
ramp always starts at the same temperature and has clearly started ramping. Note the initial a~bient and block temperatures.
do while (coolant temperature < 12~C) {

wait 1 ~econd }

{blk delta} - block temperature {h20 delta} ~ coolant temperature For the next two minutes, while the coolAnt temperature is ramping to 18~C, sum the coolant temperature and the difference between the ambient and coolant temperatures.

{temp sum} - 0 {cool sum} e 0 CA 022660l0 l999-04-Ol for (count 1 to 128 seconds) {

(32) {cool_-um} - cool_temp_-um + coolant temperature.
5 (33) {temp sum} - ambient - coolant temperature wait 1 ~econd count ~ count + 1 }

Calculate the change in temperatures over th- two ~inute 10 period.

(34) {blk delta} - block temperature - {blk delta}
(35) {h20 delta} - coolant temperature - {h20 delta}

Compute RChill, i.e., the rate of change of chiller power with coolant temperature over tbe coolant range of 10~C
to 20-C. Note that unit~ are in watt~/lO-C.

(36) Kchill ~ (Chiller Pwr ~ 18-C - Chiller Pwr ~ 10~C) Compute Sc which i~ the ~lope of tbe ramp cooling conductivity ver~us the temperature range of 18~C to lO-C. The unit~ are in watt~/10-C/10-C.

20 (37) Sc - (Kc 18 - Rc 10) / 8 Compute Rc 0, tbe ramp cooling conductance extrapolated to O-C.

(38) Rc 0 ~ Kc 10 - (Sc ~ 10) Compute Cp Cool, tbe Cp of the coolant by:

25 (39) Cp Cool ~ ( HEATPOWER ~ 128 + PUMP PWR ~ 128 - Power ~ 0~C ~ 128 - Block Cp * blk delta + K A~B ~ temp cum - Kchill ~ cool t-mp ~um 5 h20 delta where HEA~r~W~ 500 W, the heat-r power appliod to warm th- block, thu- heating the coolant It iB multiplied by 128, ac the heating interval was 128 secs PU~P PWR - 12 W, the power of the pump that circulates the coolant multiplied by 128 ceconds Pwr 0~C ~ The chiller power at O C multiplied by 128 ~econd~
Block Cp - Thermal capacity of cample block blk delta - Change in block temp over the heating interval R AMB - 20 Watt~/K, thermal conductance from cooling to ambient temp cum ~ The cum once per rec~n~ of a~bient -coolant temperature over the ~nterval h20 delta e Change in coolant temperature over interval of h-ating (approximately 6 C) Kchill ~ Slope of chiller power vercus coolant te~p.
~ool cum - The cum of coolant t-mp, once per ~econd, over the h-ating inter~al.

P: ~1~\111 l\lSl~P\,~PPL~ .ItCF
F~bru~ry ~, 1991 ~ b~ ) ~AT. TT~E OPF~ATING SYST~ - C~FTIN

CRETI~ is a ~tand alone, multitasking kernel that provides system services to other software modules called tasks. Tasks are written in the ~C" language with some time 5 critical areas written in Intel 8085 assembler. Each task has a priority level and provides an in~ep~n~ent function.
CRETIN re6ide~ in low memory and runs after th- ~tartup diagnostic~ have 6uccessfully been ex~cuted.

CRETIN handles the ta~k scheduling and allow- only one 10 task to run at a time. CRETIN receives all hardware interrupts thus enabling waiting ta~ks to run when the proper interrupt is received. CRETIN provides a real time clock to allow tasks to wait for timed events or pause for known intervals. CRETIN al-o provides intertask 15 communication through a cy~tem of mes6age nodes.

The firmware i6 compo~ed of nine task6 which are briefly described in priority order below. Subsequent section6 will describe each task in greater detail.

1. The control task (PID) i~ rcspon~ible for controlling th- ~~mple block temperature.

2. The k-yboard task i~ ~ pGn~ible for processing keyboard input from the k-ypad.

3. The timer task waits for a half ~econd hardware interrupt, then 6ends a wake up mes~age to both the ~equence and the display ta~k.

4. The ~eguence task executes the u~er ~o~am~.

5. The pause task handles programmed and keypad pauses when a program is running 6 The di6play task updates the di~play in real time 7 The printer task handles the RS-232 port communication and printing 5 8 The LED task is responsible for driving the heating LED It i~ o u-ed to cGl~ol the coolant temperature while executing In~tall 9 The link task ~tart~ files that ar- linked together in a method by ~imulating a keystroke Block TemDerature Control Program (PID Task) The Proportional Integral Differential (PID) task is responsible for controlling the absolute sample block temperature to 0.1~C, as well as controlling the sample 5 block temperature non-uniformity (TNU, defined as the temperature of the hottest well minus the temperature of the coldest well) to less than + 0.5~C by applying more heating power to the perimeter of the block to compensate for losses through the guard band edges. The PlD task is also 10 responsible for controlling the temperature of the heated cover to a less accurate degree. This task runs 5 times per ,r second and has the highest priority.

The amount of heating or cooling power delivered to the 15 sample block is derived from the difference or "error"
between the user specified sample temperature stored in memory, called the setpoint, and the current calculated sample temperature. This scheme follows the standard loop control practice. In addition to a power contribution to 20 the film heaters directly proportional to the current error, i.e., the proportional component, (setpoint temperature minus sample block temperature), the calculated power also incorporates an integral term that serves to close out any static error (Setpoint temperature - Elock temperature less 25 than o.s~C). This component is called the integral component. To avoid integral term accumulation or ~wind-up", contributions to the integral are restricted to a small band around the setpoint temperature. The proportional and integral component gains have been carefully ~elected and 30 tested, as the time constants associated with the block sensor and sample tube severely restrict the system's phase margin, thus creating a potential for loop instabilities.
The proportional term gain is P in Equation (46) below and the integral term gain is Ki in Equation (48) below.

The PID task uses a "controlled over~hoot algorithm"
where the block temperature often overshoots its final steady ~tate value in order for the sample temperature to arrive at its desired temperature as rapidly as possible.
5 The use of the overshoot algorithm causes the block temperature to overshoot in a controlled manner but does not cause the sample temperature to overshoot. Thi6 saves power and is believed to be new in PCR instrumentation.

The total power delivered to all heater of the sample 10 block to achieve a desired ramp rate is given by:

(40) Power ~ (CP / ramp rate) + bias where:
CP = Thermal mass of block bias = bias or control cooling power ramp rate - Ttj~, - T,njtj" / desired ramp rate This power is clamped to a maximum of 500 watts of heating power for safety.

With every iteration of the task (every 200ms) the system applies heating or ramp cooling power (if necessary) 20 based on the following algorithms.

The control system is driven by the calculated sample temperature. The ~ample temperature is defined as the average temperature of the li~uid in a thin walled plastic sample tube placed in one of the wells of the sample block 25 (herafter the ~block"). The time constant of the system (sample tube and its contents) i6 a function of the tube type and volume. At the start of a run, the user enters the tube type and the amount of reaction volume. The system computes a resultant time constant (I or tau). For the MicroAmpTM tube and lOO microliters of reaction volume, tau is approximately 9 seconds.

(41) Tblk-new = Tblk + Power * (200ms / CP) (42) Tsamp-new = Tsamp + (Tblk-new - Tsamp) * 200 ms / tau where:
Tblknew = Current block temperature Tblk - - = Block temperature 20Oms ago Power = Power applied to block CP = Thermal mass of block Tsampnew = Current sample temperature Tsamp = Sample temperature 20Oms ago Tau = Thermal Time Constant of sample tube, adjusted for sensor lag (approximately 1.5) The error signal or temperature is simply:
(43) error = Setpoint - Tsamp-new As in any closed loop system, a corrective action (heating or cooling power) is applied to close out part of the current error. In Equation (45) below, F is the fraction of the error signal to be closed out in one sample period (200mS).

(44) Tsamp-ne~ = Tsamp + F * (SP - Tsamp) where SP = the user setpoint temperature Due to the large lag in the system (long tube time constant), the fraction F is set low.

Combining formulas (42) and (44) yields:

(45) Tsamp-new = Tsamp + (Tblk-new~Tsamp) * .2 / tau = Tsamp + F *

(SP~Tsamp) Combining formulas (41) and (45) and adding a term P (the proportional term gain) to limit block temperature oscillations and improve system stability yields:

(46) Pwr = CP * P/T * ((SP - Tsamp) * F * tau/T + Tsamp - Tblk) where P = the proportional term gain and-T = the sample period of 0.2 seconds (200 msec).
and P/T = 1 in the preferred embodiment Equation (46) is a theoretical equation which gives the power (Pwr) needed to move the block temperature to some desired value without accounting for losses to the ambient through the guardbands, etc.
Once the power needed to drive the block is determined via Equation ~46), this power is divided up into the power to be delivered to each of the three heater zones by the areas of these zones. Then the losses to the manifolds are determined and a power term having a magnitude sufficient to compensate for these losses is added to the amount of power to be delivered to the manifold heater zone. Likewise, another power term sufficient to compensate for power lost to the block support pins, the block temperature sensor and the ambient is added to the power to be delivered to the edge heater zones. These additional terms and the division of power by the area of the zones convert Equation (46) to Equations (3), (4) and (5) given above.
Equation (46) is the formula used by the preferred embodiment of the control system to determine the required heating or cooling power to the sample block.
When the computed sample temperature is within the "integral band", i.e., _ 0.5~C around the target temperature (SP), the gain of the proportional term is too small to close out the remaining error. Therefore an integral term is added to the proportional term to close out small errors. The integral term is disabled outside the integral band to prevent a large error signal from accumulating. The algorithm inside the "integral band" is as follows:

(47) Int_sum (new) = Int_sum (old) + (SP - Tqamp) (48) pwr_adj=Ki * Int_sum (new) where, Int_sum = the sum of the sample period of the difference between the SP and Ts~p temperature, and Ki = the integral gain (512) in the preferred embodiment.

Once a heating power has been calculated, the control software distributes the power to the three film heater zones 254, 262 and 256 in Figure 13 based on area in the preferred embodiment. The edge heaters receive additional power based upon the difference between the block temperature and ambient temperature. Similarly; the manifold heaters receive additional power based upon the difference between the block temperature and the coolant temperature.

PID Pseudocode Upon System Power up or Reset Turn off ramp cooling Turn off all heaters Calculate heater resistances Do Forever - executes every 200 ms If (block temperature ~ 105) then Turn off heaters Turn on ramp valves Display error message Read the line voltage {linevolts}

Read the coolant sensor and convert to temperature {h20temp}
Read the ambient sensor and convert to temperature {ambtemp}.
Read the heated cover sensor and convert to temperature {cvrtemp}
Read the sample block sensor and convert to temperature {blktemp}. This portion of the code also reads the temperature stable voltage reference and compares the voltage to a reference voltage that was determined during calibration of the instrument.
If there is any discrepancy, the electronics have drifted and the voltage readings from the temperature sensors are adjusted accordingly to obtain accurate temperature readings.

Compute the sample temperature {tubetenths~ or the temperature that gets displayed by using a low-pass digital filter.

(49) tubetenths = TTn1 + (TBn - TTn1) * T/tau where TTnl = last sample temp {tubetenths}
TBn = current block sensor temp {blktenths}
T = sample interval in seconds = 200ms tau = tau tube ~cf_tau} - tau sensor {cf_lag}

Equation (49) represents the first terms of a Taylor series expansion of the exponential that defines the calculated sample temperature given as Equation (6) above.

Compute the temperature of the foam backing underneath the sample block, {phantenths} known as the phantom mass. The temperature of the phantom mass is used to adjust the power delivered to the block to account for heat flow in and out of the phantom mass. The temperature is computed by using a low pass digital filter implemented in software.

(50) phantenths = TTn1 + (TBn - TTn1) * T/tau where TTnl = Last phantom mass temp (phantenths) TBn = Current block sensor temp {blktenths}
T = Sample interval in seconds = 20Oms taufOam = Tau of foam block = 30 secs.

Compute the sample temperature error (the difference between the sample temperature and the setpoint temperature) {abs tube err}.

Determine ramp direction {fast ramp} - UP RAMP or DN RAMP

If (sample temperature is within ERR of cetpoint (SP)) then PID not in fast transition mode. {fast ramp} ~ OFF
where ERR ~ the temperature width of the ~integral band~, i.e., the error band ~urrounding the target or ~etpoint temperature.

Calculate current control cooling power {cool_ctrl} to determine how much heat i~ being lost to the bias cooling channel~.
Calculate current ramp cooling power lcool ramp}

Calculate {cool brkpt}. {cool brkpt} is a cooling power that is used to determine when to make a transition from ramp to control cooling on downward ramp~. It is a function of bloek and coolant temperature.

The control cooling power {cool ctrl} and the ramp cooling power {cool ramp} are all factors which the CPU must know to control downward temperature ramps, i.e., to calculate how 25 long to keep the ramp cooling solenoid operated valves open.
The control cooling power i~ egual to a constant plus the temperature of the coolant times the thermal conductance from the block to the bias cooling channels. Likewi-e, the ramp cooling power is equal to the difference between the block 30 temperature and the coolant temperature times the thermal _ - 130 -conductance from the block to the ramp cooling channels.
The cooling breakpoint is equal to a constant times the difference in temperature between the block and the coolant.
Calculate a heating or cooling power (int_pwr) needed to move the block temperature from its current temperature to the desired setpoint (SP) temperature.

(51) (int_pwr) = KP * CP * [(SP - Ts~p) * {cf_kd} +
T S -- TBLK ]

where:
KP = Proportional gain - P/T in Equation (46) = approximately one in the preferred embodiment CP = Thermal mass of block SP = Temperature setpoint Ts~ = Sample temperature T3L~ = Block temperature cf_kd = Tau * Kd / Delta_t where tau is the same tau as used in Equation (49) and Kd is a constant and Delta_t is the 200 msec sample period.

If (sample temperature is within {cf_iband} of setpoint) then integrate sample error {i_sum}
else (52) clear {i_sum = 0}.

Calculate the integral term power.
(53) integral term = {i_sum} * constant {cf_term}.

Add the integral term to the power.
(54) {int_pwr} = {int_pwr} + integral term Adjust power to compensate for heating load due to the effects of the phantom mass (foam backing) by first finding the phantom mass power then adding it to power (int_pwr).

Calculate phantom mass power {phant_pwr} by:
(55) phant_pwr = C * (blktenths - phantenths) / 10 where: C = thermal mass of foam backing (1.0 W/K) Adjust heater power {int_pwr} = {int_pwr} + {phant_pwr}

Compute power needed in manifold heaters ~auxl_power}
which will compensate for loss from the sample block into the manifold edges that have coolant flowing through it. Note that if the system is in a downward ramp, {auxl_power} = O. The manifold zone power required is described below:

(57) {auxl_poWer} = K1* (TBLK ~ T~B) + K2 (TBLK TCOOL) +
K5*(dT/dt) where:
K1 = Coefficient {cf_lcoeff}
.. K2 = Coefficient {.cf_2coeff}
K5 = Coefficient {cf_5coeff}
dT/dt = Ramp rate TBLK = Block temperature T~B = Ambient temperature TCOOL = Coola-nt temperature Compute power needed in edge heaters {aux2 power} which will compensate for losses from the edges of the sample block to ambient. Note that if we are in a downward ramp {aux2_power} = O. The edge zone power required is described below:

.

(58) {aux2_power} = K3* (T~L~ ~ TAMR) + K4~ (TtL~ ~ T~~L) +
K6*(dT/dt) where:
K3 = Coefficient {cf_3coeff}
K4 = Coefficient {cf 4coeff}
K6 = Coefficient {cf 6coeff}
dT/dt ~ Ramp rate TBL~ ~ Block temperature T~p e A~bient temperature TU~L c Coolant temperature Delete contribution of manifold {auxl power} and edge heater power {aux2 power} to obtain total power that must be supplied by main heaters and coolers.
~5 (59) {int pwr} = {int power} - {auxl power} -{aux2 power}

Decide if the ramp cooling should be applied. Note that {cool brkpt} is used as a breakpoint from ramp cooling to control cooling.

If (int pwr < cool brkpt and performing downward ramp) to decide whether block temperature i6 SO much higher than the setpoint temperature that ramp cooling is needed then Turn ON ramp valves else Turn OFF ramp valves and depend upon bias cooling At this point, {int pwr} contains the total heater power and ~auxl power} and {aux2 power} contain the loss from the block out to the edges. The power supplied to the auxiliary heaters is composed of two components: aux power and 30 int_power. The power is distributed {int_pwr} to the main and auxiliary heaters based on area.
total_pwr = int_pwr int pwr = total_pwr ~ 66%
auxl_power = total pwr * 20%+ auxl_power aux2 power = total_pwr * 14S+ aux2_power Compute the n~mher of half cycles for the triac to conduct 5 for each end zone and each iteration of the control loop to send the appropriate amount of power to the heaters. ~his loop executes once every 1/5 second, therefore there are 120/5 ~ 24 half cycles at 60Hz or 100/5 - 20 at 50Hz. The number of half cycles is a function of requested power 10 {int pwr}, the current line voltage {linevolts} and the heater resistance. Since the exact power needed may not be delivered each loop, a remainder is calculated {delta power}
to keep track of what to include from the last loop.

(60) int pwr ~ int pwr + delta power 15 Calculate the number of 1/2 cycles to keep the triac on.
Index is equal to the number of cycles to keep the triac on.

(61) index ~ power * main heater ohms * [20 or 24] /
linevolts squared where Equation (61) is performed once for each heater zone and where "powern ~ int pwr for the main 20 heater zone, auxl_pwr for the manifold heater zone and aux2 pwr for the edge heater zone.

Calculate the amount of actual power delivered.

(62~ actual power ~ linevolts ~quared * index / main heater resistance 25 Calculate the remainder to be added next time.

(63) delta_power = int_pwr - actual_power Calculate the number of 1/2 cycles for the ~dge and manifold heaters using the ~ame technique described for the main heaters by sub6tituting {auxl pwr} and ~aux2 pwr} into Equation (60) 5 Load the calculated counts into the counters that control the main, manifold and edge triacs Look at h-ated cover c-nsor If h-at-d cover i~ 8 than 100~C, then load heated cover counter to ~upply 50 w~tts of power 10 Look at sample temperature If it i6 greater than 50~C, turn on HOT LED to warn user not to touch block END OF FOREVER LOOP

Xeyboard Task The purpose of the keyboard task i- to wait for the user to press a key on the keypad, compare the key to a list of valid keystrokes for the current etate, execute the command 5 function associated with the valid key and change to a new state. Invalid keystrokes are indicated with a beep and then ignored. This task is the heart of the etate driven user interface. ~t ie ~-tate driven" because the action taken depende on the current etate of the ueer interface.

10 KeYboard Task Pseudocode:
Initialize keyboard task variables.
Turn off the cursor.
If (install flag not ~et) then Run the install program.
15 Send a message to pid task to turn on the heated cover.
If (the power failed while the ueer was rl~nning a program) then Compute and display the number of minutes the power was off for.
Write a power failure status record to the history file.
Send a message to the eeguence taek to etart a 4~C soak.
Give the user the option of reviewing the history file.
If (the user reguest to review the hietory file) then Go to the history file display.
25 Dieplay the top level ccreen.

Do ~orever Send a message to the syetem that this taek is waiting for a hardware interrupt from the keypad.
Go to ~leep until thie interrupt i6 received.
When awakened, read and decode the key from the keypad.
Get a list of the valid keye for the current ~tate.
Compare the key to the liet of valid keye.
If (the key is valid for this etate) then Get the "a~tion" and next etate information for this key.
Execute the ~action" (a co~mand function) for this state.
Go to the next state.
5 Else Beep the beeper for an invalid key.
End of Forever Loop - 13~ -Timer Task Overview The purpose of the timer task i~ to wake up the ~equence and the real time display task every half a ~econd The timer task asks the ~y~tem (CRETIN) to wake it up whenever 5 the half second hardware interrupt that is generated by the clock/calendar device is received The timer task then in turn ~ends 2 wake up messages to the sequence ta6k and the real time display task respectively This inter~ediate task is necessary ~ince CRETIN will only service one task per 10 interrupt and thus only the higher priority task ~the seguence task) would execute Timer Task Pseudocode Do Forever Send a message to the system that this task is waiting for a hardware interrupt from the clock/calendar device Go to ~leep until this interrupt is received When awakened, send a mes~age to the sequence and to the real time display task End Forever Loop P~ \p\A~pL~ cF

Seauence Task Overview The purpo6e of the ~eguence ta~k i~ to execute the contents of a user defined program. It 6equentially steps through each 6etpoint in a cycle, consisting of a ramp and 5 a hold cegment, and sends out setpoint temperature messages to the pid task which in turn controls the temperature of the 6ample block. At the end of each 6egment, it ~ends a message to the real time display task to ~witch the display and a message to the printer task to print the ~egment's 10 runtime information. The user can pause a running program by pressing the PAUSE key on the keypad then resume the program by pressing the START key. The user can prematurely abort a program by pressing the STOP key. This task executes every half a ~econd when it is awakened by the 15 timer task.

Seouence Task Pseudocode:
Do Forever Initialize sequence task variables.
Wait for a message from the keyboard task that the user has 20 pressed the START key or celected START from the menu or a message from link task that the next program in a method is ready to run.
Go to 61eep until thi~ message i6 received.
When awakened, update the ADC calibration reading~ to account 25 for any drift in the analog circuitry.
If (not starting the 4~C power failure ~oak ~equence) then Send a me~sage to the printer task to print the PE title line, system time and date, program configuration parameters, the program type and its number.

30 If (starting a HOLD program) then Get the temperature to hold at {hold tp}.
Get the number of ~econds to hold for {hold time}.
If (ramping down more than 3-C and {hold tp} > 45~C) then Post an intermediate setpoint.
Else Post the final 6etpoint ~hold tp}.
While (counting down the hold time {hold time}) Wait for half ~econd wake up message from timer task.

Check block ~ensor for open or short.
If (keyboard task detected a PAUSE key) then Post a ~etpoint of ~L~ ~nt ~a~ple temp.
Send a message to wake up the pause task.
Go to sleep until awakened by the pause task.
Post pre-pause setpoint.
If (an intermediate setpoint was posted) then Post the final setpoint.
If (the ~etpoint temp is below ambient temp and will be there for more than 4 min.) then Set a flag to tell pid task to turn off the heated cover.
Increment the half ~econd hold time counter 20 {store time}.
Post the final ~etpoint again in case the hold time expired before the intermediate 6etpoint was reached - this insures the correct ~etpoint will be written the hictory file.
Write a data record to the hi6tory file.
Send a me~6age to the printer ta~k to print the HOLD
info.
End of HOLD program Else if (6tarting a CYCLE program) then Add up the total number of ~econds in a cycle {secs in run~, taking into account the in~trument ramp time and the user programmed ramp and hold time6.
Get the total number of 6econds in the program by multiplying the number of seconds in a cycle by the number of cycles in a program {num cyc}.
Total {secs_in run} - {sec~ in run} per cycle ~ {num cyc}.
While (counting down the number of cycles {num cyc}) While (counting down the number of setpoints {num seg}) Get the ramp time {ramp time}.
Get the final setpoint temp {t final}.
Get the hold time {local time}.
Send a mes6age to the real time display task to display the ramp segment information.
If (the user programmed a ramp time) then Compute the error {ramp err} between the programmed ramp time and the actual ramp time as follows. Thi6 eguation is based on empirical data.

{ramp err} ~ prog ramp rate ~ 15 + 0.5 (up ramp) {ramp err} - prog ramp rate ~ 6 + 1.0 (down ramp) where:
prog ramp rate - (ab6(T~ - Tc) - 1) / {ramp time}

T~ ~ setpoint temp {t final}
Tc - current block temp {blktemp}
abs - absolute value of the expression Note: the '- 1' is there becau6e the clock starts within l-C of ~etpoint.

new ramp time - old {ramp time} - ~ramp err}
If (new ramp time ~ old {ramp time}) then new ramp time e old {ramp time}.
Else . .

new ramp time ~ O
While (sample temp is not within a user configured temp {cf clk dev} of setpoint) s Wait for half second wake up message from timer task Post a new ramp ~etpoint every second Else if (ramping down more than 3 C and ~t final~
>
45 C) then Post an intermediate ~etpoint While (sample temp i8 not within a user configured temp {cf clk dev} of ~etpoint) Wait for half second wake up message from timer task Increment the half ~econd ramp time counter Check block ~ensor for open or short If (keyboard task detected a PAUSE key) then Post a ~etpoint of current 6ample temp Send a message to wake up the pause task Go to ~leep until awakened by the pause task Post pre-pau6e ~etpoint Post the final setpoint While (sample temp is not within a user configured temp {cf clk dev} of setpoint) Wait for half cecond wake up message from timer task Increment the half second ramp time counter Check block sensor for open or short If (keyboard task detected a PAUSE key) then Post a setpoint of current sample temp.
Send a message to wake up the pause task.
Go to ~leep until awakened by the pause task.
Post pre-pause ~etpoint.
Send a message to the printer task to print the ramp information.
Beep beeper to ~ignal ~nd of ramp ~egment.
Send a message to the real time di6play task to display the ramp segment information.
While (counting down the hold time) Wait for half 6econd wake up message from timer task.
Increment the half second hold time counter.
Check block sen60r for open or short.
If (keyboard task detected a PAUSE key) then Post a setpoint of current ~ample temp.
Send a message to wake up the pause task.
Go to ~leep until awakened by the pause ta6k.
Post pre-pause ~etpoint.
Write a data record to the history file.
Send a mes6age to the printer task to print the hold information.
If (the final 6etpoint temp has drifted more than the user configurable amount {cf temp dev}) then Write an error record to the hi~tory file.
Check for a programmed pau6e.
Go to next 6egment.
Send a message to the printer ta~k to print an end of cycle message.
Go to next cycle.
3S End of CYCLE program.

Else if (starting an AUTO-CYCLE program) then Add up the total number of seconds in each program {secs in run} taking into account the instrument ramp time and the user programmed hold times which can be automatically incremented or decremented by a programmed amount each cycle.
While (counting down the number of cycles {num cyc}) While (counting down the number of ~etpoints {num_seg}) Get the final ~etpoint temp {t final}.
Get the hold time {time hold}.
Check if the user programmed an auto increment or decrement of the setpoint temp and/or the hold time and adjust them accordingly.
If (the auto increment or decrement of the temp causes the setpoint to go below O'C or above 99.9~C) then An error record is written to the history file.
~he setpoint is capped at either O-C or 99.9~C.
Send a message to real time display task to display the ramp ~egment information.
If (ramping down more than 3~C and {t final} >
45~C) then Post an intermediate ~etpoint.
While (sample temp is not within a user configured temp {cf clk dev} of ~etpo~nt) Wait for half ~econd wake up message from timer t~sk.
Increment the half ~econd ramp time counter.
Check block ~ensor for open or short.
If (keyboard task detected a PAUSE key) then .

Post a ~etpoint of current 6ample temp.
Send a message to wake up the pause task.
Go to sleep until awakened by the pause task.
Post pre-pause 6etpoint.
Post the final ~etpoint.
While (sample temp is not within a user configured temp {cf clk dev} of setpoint) Wait for half second wake up message from timer task.
Increment the half ~econd ramp time counter.
Check block ~ensor for open or short.
If (keyboard task detected a PAUSE key) then Post a ~etpoint of current sample temp.
Send a message to wake up the pause task.
Go to sleep until awakened by the pause task.
Post pre-pause setpoint.
Send a message to the printer task to print the r~mp segment information.
~eep beeper to signal end of ramp portion of ~egment.
Send a message to the real time di6play task to di~play the hold segment information.
While (counting down the hold time) Wait for half second wake up mes6age from timer task.
Increment the half second hold time counter.
Check block sensor for open or ~hort.
If (keyboard ta~k detected a PAUSE key) then Post a setpoint of current 6ampl- temp.
Send a message to wake up the pause task.
Go to sleep until awakened by the pause task.
Post pre-pause 6etpoint.
Write a data record to the history file.
Send a message to the printer task to print the hold information.
If (the final ~etpoint temp has drifted more than the user configurable amount {cf temp dev}) then Write an error record to the ~i~tory file.
Go to next segment.
Send a message to the printer task to print an end of cycle message.
Go to next cycle.
End of AUTO-CYCLE program.

15 Else if (starting a POWER FAILURE ~eguence) then Post a setpoint of 4 ~C.
Set a flag ~subamb hold} so that the pid task will 6hut off the heated cover.
DO FOREVER
Wait for a half second wake up message from the timer task.
Increment the half ~econd hold time counter.
END FOREVER LOOP
End of power failure ~equence 25 Write a run end status record to the hi~tory file.
If (running a method) Set a flag {weird flag} 80 the link ta~k will know to send a message to the sequence task to ~tart the next program running.
30 Else Return user interface to idle ~tate di~pl~y.
End of Forever Loop Pause Task Overview The purpose of the pause task is to handle either a pause that the user programs in a CYCLE program or a pause when the user presses the PAUSE key on the keypad.
When the seguence task encounters a programmed pause while executing a CYCLE program, it goes to sleep and awakens the pause task. The pause task in turn ~ends a message to the real time display task to continually display and decrement the time the user asked to paus- for. When the pau~e timer 10 times out, the pause task ~ends a me6sage to awaken the sequence task and then goes to sleep. The user can prematurely resume the program by pressing the START key on the keypad or can prematurely abort the program by pressing the STOP key.
When the keyboard task detects a PAUSE key while a program is running, it 6ets a flag {pau~e flag} then waits-for the sequence task to acknowledge it. When the 6equence task sees this flag set, it sends an acknowledgment message back to the keyboard task then puts it6elf to ~leep. When the 20 keyboard task receives this message, it awakens the pause task. The pause task ~ends a ~essage to the real time display task to continually di~play and increment the amount of time the program is paused for. The timer will time out when it reaches the pause time limit set by the user in the 25 configuration ~ection. The u6er can re~ume the program by pressing the START key on the keypad or abort the program by pressing the STOP key.

Pause Task Pseudocode:
Do Forever Wait for a message from the keyboard task indicating a keypad pause, or a message form the 6equence task indicating a user programmed pause.
Go to sleep until a mes~age is r-ceived.
When awakened, check a flag for the type of pause initiated.

If (it is a programmed pause) then Send a message to the real time display task to display the pause timer counting up.
Else Send a message to the real time display task to display the pause timer counting down.
While (counting down the time out counter) Send a message to the system to suspend this task for half a second.
Send a mescage to the printer task to print the pause information.
If (it is a progra~med pause) then The pause has timed out so send a message to the wake up the sequence task.
Send a message to the real time display task to halt the pause display.
Send ~ message to the real time display task to resume the running program display.
Else (it is ~ keypad pause) The pause has timed out and the program must be aborted so send a message to the system to halt the sequence task ~nd send it back to the top of its FOREVER loop.
If (the program running was a HOLD program) Send ~ message to the printer task to print the hold information.
Write a status record to the history file.
Return the user interface to it~ idle state.
Display ~n ~bort message.
30 End of Forever Loop CA 022660l0 l999-04-Ol Dis~laY Task Overview The purpose of the real time display task is to di~play temperatures, timers, sensor readings, ADC channel readings, and other parameters that need to be continually updated 5 every half second.

Dis~lay Task Pseudocode:
Initialize display task variables.

Do Forever Wait for a ~essage every half 6econd from the timer task.
Go to sleep until the message is received.
When awakened, check if another task has ~ent a list of parameters to display or a flag to halt the current update.
Toggle the half second flag {half ~ec}.
If (there's a list of parameters to display) then Set a semaphore 80 no one else will update the display.
Turn off the cursor.
While (etepping through the li~t of parameters) If (it i6 a time parameter) then Di6play the time.
If (half 6econd flag {half ~ec} is set) then Increment or decrement the time variable.
El6e if (it i6 a decimal number) then Di~play a d-cimal nu~ber.
Else if (it i8 an integer number) then Display the integer.
Else if (it iB an ADC ch~nnel readout) then Read the counts from the ADC channel.
If (need it displayed as mV) then Convert counts to mV.
Display the value.
Else if (it is a power dieplay) then Display the power in terms of watts.

Else if (it i6 the hours left parameter) then Convert ceconds to tenths of hours.
Di~play the hours left $n tenths of hour~.
If (half cecon~ flag {half sec} is ~et) then Decrement the ~econ~s variable.
If (the cur60r was on) then Turn it back on.
Store the current ~ystem time in battery RAM.
Clear the ~emaphore to release the display.
10 End of Forever-Loop CA 022660l0 l999-04-Ol Printer Task Overview The purpose of the printer taek ie to handle the runtime printing. It is a low priority task and should not interfere with other time critical tasks.

S Printer Task Pseudocode:
Do Forever Wait for a message from another task that wiehes to print.
Go to sleep until a message is received.
When awaken, make local copies of the global variables to be printed.
Post a printer acknowledgement meesage.
If (need to print a ~tatus or error ~e6sage) then Print the information contained in the current hietory record.
Else if (need to print the page header) then Print the company name, instrument ID, firmware ver~ion number and the current ~ystem time and date.
Else if (need to print the program header) then Print the type of progr~m and it~ number.
Else if (need to print the program configuration parameters) then Print the tube type, reaction volume and the sample temperature deviation from ~etpoint that ~tarts the clock.
Elee if (need to print ~nd of cycle information) then Print the ending time and temperature.
Else if (need to print ~egment information) then Print either the ramp or hold ~egment information.
Else if (need to print a pau~e ~tatus ~es~age) then Print the ~mount of time pau~ed for and at what temp.
End of Eorever Loop nFn Task overview The purpose of the LED task is to make the illumination of the "Heating" LED reflect the power applied to the main beater. This is a low priority task that runs once a second.

T.Fn Task Pseudocode:
5 Initialize LED task variables.

Do Forever Send a message to the system to wake this task every ~econd.
Go to 6leep.
When awaken, load counter 2 of PIC timer A with a value that reflect~ the power applied to the main heater as follow6:

load counter with value ~ {K htled~ * ~ht led}

Where:
~ htled} holds a constant to compute the time to pul6e the heating LED and i6 egual to 15200 / 500.
15200 i6 a little greater than the PIC's clock of 14.4XHz and thi6 iB the value loaded into the timer to keep the LED constantly on. 500 is the main heater power.

~ht_led} will be a valu- between 0 and 500 and will be egual to the watt- applied to the ~ain heater.
End of Forever Loop nink Task overview 25 The purpose of the link task i~ to simulate the user pressing the START key on the keypad. This task is necessary 60 that programs can be executed one right after the other (as in a method) w$thout user intervention. The link task wakes up the seguence task and it begins running the next program as if the START key were pressed.

!ink T~sk Pseudocode:
Initi~lize link task vari~bles.

Do Forever If (the flag {weird flag~ is cet ~nd it ic not the first file in the method) then Send a message to the seguence task to wake up.
End of Forever Loop Start Up Sequence PO~ER-~P 8~Q~NC~

When the power to the instrument is turned on or the software does a RESET, the following sequence takes place.
5 Note: the numbers below correspond to numbers on the flow chart.

1. Transmit a Ctrl-G (decimal 7) character out the RS-232 printer port. Poll the RS-232 port for at least 1 second and if a Ctrl-G is received, it is assumed that an external computer i6 attached to the port and all communication during the power-up ~eguence will be redirected from the keypad to the RS-232 port. If no Ctrl-G is received, the power-up sequence continues as normal.
15 2. Check if the MORE key is depre~sed. If 60, go straight to the 6ervice-only hardware diagnostics.
3. The next 3 tests are an audio/vi~ual check and cannot report an error: 1) the beeper beeps 2) the hot, cooling, and heating LEDs on the keypad are flashed 3) each pixel of the display iB highlighted. The copyright and instrument ID ~creens are di6played as the power-up diagnostics execute.
4. Should an error occur in one of the power-up diagnostics,-the name of the component that failed is displayed and the keypad i~ locked except for the code 'MORE 999' which will gain acces6 to the 6ervice-only hardware diagno~tic~.
5. Check channel O of the PPI-B device to 6ee if the automated test bit is pulled low. If it i~, run the UART test. If the test pas6e6, beep the beeper continuously.
6. Start the CRETIN operating sy6tem which in turn will start up each task by priority level.

7 Check a flag in battery RAM to 6ee if the instrument has been calibrated If not, display an error message ~nd lock the keypad except for the code 'MORE
999' which will gain access to the ~ervice-only calibration tests 8 Run a test that measures the ~oltage and line freguency and see if both these values match the configuration plug ~elected while calibrating the instrument If not, display an error me~age and lock the keypad except for the code 'MORE 999' which will gain access to the ~ervice-only calibration te6ts 9 Perform the heater ping test as described in the Install ~ection If the heaters are wired wrong, display an error message and lock the keypad except for the code 'MORE 999' which will gain access to the service-only calibration tests Check a flag in battery RAM to cee if the instrument has been installed If not, display an error message and lock-the keypad except for the code 'MORE 999' which will gain acces6 to the in~tall routine 11 If not in remote mode, check a flag in battery RAM to see if there was a power failure while the instrument was running. If 80, 6tart a 4~C coak and di6play the amount of time the power was off for Ask the user lf they wi6h to view the hi~tory file which will tell them exactly how far along they were in the run when the power went off If thcy ~-lect ycs, they go ~traight to the user diagnostics 30 12 Beep the beeper and clear the remote mode flag 80 all communication now is back through the keypad 13 Check a flag in battery RAM to ~ee if manufacturing wants their test program automatically started If ~o, start the program running and reset the instrument after its done 14 Display the top level user interface ~creen Referring to Figure 50, there i6 6hown a cross-sectional view of a larger volume, thin walled reaction tube marketed under the trademark MAXIAMP. This tube i~ useful for PCR reactions wherein reagents or other material~ need 5 to be added to the reaction mixture which will bring the total volume to greater than 200 microliters. The larger tube shown in Figure 50 made of Himont PD701 polypropylene or Valtec KH-444 polypropylene and has a thin wall in contact with the sample ~lock. Whatever material is 10 selected should be compatible with the DNA and other components of the PCR reaction mixture oo a~ to not impair PCR reaction processing ~uch as by having the target DNA
stick to the walls and not replicate. Glas6 i6 generally not a good choice because DNA has been known to ~tick to the 15 walls of glass tubes.
The dimension A in Figure 50 is typically C.012 +
.001 inches and the wall angle relative to the longitudinal axis of the tube is typically 17~. The advantage of a 17~
wall angle is that while downward force cau~es good thermal 20 contact with the sample block, the tubes do not jam in the sample wells. The advantage of the thin wall~ i~ that it minimizes the delay between changes in temperature of the sample block and corresponding changes in temperature of the reaction mixture. This means that if the user wants the 25 reaction mixture to remain within 1~C of 94~C for 5 ~econds in the denaturation segment, and programs in these parameter6, he or she gets the 5 ~econd denaturation interval with less time lag than with conventional tubes with thicker walls. Thi~ performance characteri~tic of 30 being able to program a ~hort ~oak interval ~uch as a 5 second denaturation 60ak and get a soak at the programmed temperature for the exact programmed time is enabled by use of a calculated ~ample temperature to control the timer. In the system described herein, the timer to time an incubation 35 or soak interval is not ~tarted until the calculated sample temperature reaches the programmed soak temperature.

Further, with the thin walled sample tubes, it only takes about one-half to two-thirds a~ long for the eample mixture to get within 1~C of the target temperature as with prior art thick-walled microcentrifuge tubes ~nd this is 5 true both with the tall MAXIAMP~ tube shown in Figure 50 and the smaller thin walled MICROAMP~ tube chown in Figure 15 The wall thicknes6 of both the M~TAMP~ and MICROAMP~
tubes i8 controlled tightly in the manufacturing proce66 to be as thin as possible consistent with adeguate ~tructural 10 strength Typically, for polypropylene, thi- will be anywhere from 0 009 to 0 012 inche~ - If new, ~ore exotic material~ which are ~troger than polypropylene are u~ed to achieve the advantage of epee~Aing up the PCR reaction, the wall thickness can be less ~o long as adeguate strength is 15 maintained to withstand the downward force to as6ure good thermal contact, and other ~t~JsFes of normal use With a height (dimension B in Figure 50) of 1 12 inches and a dimension C of 0 780 inches and an upper ~ection wall thickness (dimension of D) 0 395 inches, the ~AXIAM~ tube's 20 time constant is approximately 14 second6 although this has not been precisely measured as of the time of filing The MICROAMP tube time constant for the shorter tube shown in Figure 15 is typically approximately 9 5 second6 with a tube wall thickness in the conical 6ection of 0 009 inches plus 25 or minus 0 001 inches Figure 51 ~hows the results of use of the thinn-r walled MICROAMP tube A similar spe~Ae~ up attainment of target temperature6 will result from use of the thin wall-d MAXIAMP tube Referring to Figure 51, there is shown a graph of the relative times for the calculated sample temperature in a MICROAMP tube versus the time for a prior art tube to reach a temperature within l C of a target denaturation temperature of 94 C from a starting temperature of 72 C In 35 Figure 51, a 100 microliter eample was present in each tube The curve with data points marked by open boxes is the calculated sample temperature response for a MICROAMP tube with a 9 5 6econd response time and a 0 009 inch wall thickness The curve with data point~ marked by X'~
represents the calculated cample temperature for a lO0 5 microliter sample in a prior art, thick walled microcentrifuge tube with a 0 030 inch wall thickness This graph shows that the thin walled ~ICROAMP tube oample reaches a calculated temperature within l C of the 94~C
target eoak temperature within approximately 36 ~econd~
10 while the prior art tubes take about 73 ~;sc~nd~ This iB
important because in instruments which do not ~tart their timers until the ~oak temperature i6 substantially achieved, the prior art tube6 can ~ub6tantially increase overall processing time especially when concidered in light of the 15 fact that each PCR cycle will have at least two ramp6 and 60aks and there are generally very many cycles performed Doubling the ramp time for each ramp by using prior art tubes can therefore dra~tically increase proce6sing time In sy6tems which start their time~ based upon 20 block/bath/oven temperature without regard to actual ~ample temperature, the6e long delays between changes in block/bath/oven temperature and corresponding changes in sample mixture temperature can have oerious negative conseguences The problem i6 that the long delay can cut 25 into the time that the reaction mixture i~ actually at the temperature programmed for a soak For very short ~oak~ as are popular in the late~t PCR p~c~e6se~, th- r-action mixture may never actually r-ach the ~G~lammed ~oak temperature before the heating/cooling ~ystem starts 30 attempting to change the reaction mixture temperature Figure 50 ~hows a polypropylene cap 650 connected to the MAXIAMP ~ample tube by a pla~tic web 652 The outoide diameter E of the cap and the in-ide diameter F of the tube upper ~ection are si2ed for an interference fit of between 35 0 002 and 0 005 inches The inside surface 654 of the tube should be free of flash, nicks and scratches ~o that a gas-tight seal with the cap can be formed.
Figure 52 shows a plan view of the tube 651, the cap 650 and the web 652. A shoulder 656 prevents the cap from being pushed too far down into the tube and allows 5 sufficient projection of the cap above the top edge of the sample tube for making contact with the heated platen. This also allows sufficient cap deformation such that the minimum acceptable force F in Figure 15 can be applied by deformation of the cap.
In the preferred embodiment, the tube and cap are made of Himont PD701 polypropylene which is autoclavable at temperatures up to 126~C for times up to 15 minutes. This allows the disposable tubes to be sterilized before use.
Since the caps are permanently deformed in use in machines 15 with heated platens, the tubes are designed for use only once.
Caps for the MICROAMP tubes are available in connected strips of 8 or 12 caps with each cap numbered or as individual caps. Single rows of caps may be used and the 20 rows may be easily shortened to as few as desired or individual caps may be cut off the strip. Caps for MAXIAMP
tubes are either attached as shown in Figure 50, or are separate individual caps.
The maximum volume for post-PCR reagent additions to 25 permit mixing on the MICROAMP tube is 200 microliters and is up to 500 microliters for the MAXIAMP tube. Temperature limits are -70~C to.126~C.
The response time depends upon the volume of the ~ample. Response is measured as the time for the ~ample to 30 come within 37% of the new temperature when the block suddenly changes temperature. Typical response time for a 50 microliter fill are 7.0 seconds and for a 20 microliter fill are 5.0 seconds.

APPENDIX A
User Interface The objective of the GeneAmp PCR System 9600 ueer interface is to provide a simple way to develop and run 5 programs that perform PCR.

There are 3 types of p~G~,ams available. The UOLD ~G~Lam consists of a single setpoint held for a ~et amount of time or held for an infinite amount of time ~nd terminated by the STOP key. The CYCLE program add~ the features of timed 10 ramps and programmable pauses. This program allows up to 9 setpoints and up to 99 cycles. The A~O program allows the user to increment or decrement the setpoint time and/or temperature a fixed amount every cycle. This program also allows up to 9 cetpoints and up to 99 cycles. A ~E~OD
15 program provides a way to link up to 17 hold, cycle or auto programs together.

A total of 150 programs can be stored with numbers ranging from l to 150. Programs can be created, stored, protected, printed, or deleted. A directory of the stored programs can 20 be viewed or printed.

~E SY8TEM 9600 ~EYPAD

Heating Cooling PAUSE OPTION l 2 3 Hot 25 RUN starts a program running from the program display or restarts a programmed or keypad pause.
MORE toggles the runtime displays and also accesses the service-only functions (if followed ~y the code ~ 999).
30 BACK moves to the previous field within the same screen. If currently positioned on the first field, it moves to the previous wreen.
STEP moves down to the first field in the next screen.
PAUSE starts a paused time-out for manual interruptions.
~5 OPTION either moves the cursor left-to-right through the menu items (rolling over to the leftmost option) or toggles the YES/NO response.
STOP aborts a running program or moves the user up one level in the user interface.
40 CE clears invalid numeric entries.
ENTER accepts the current numeric entry, accepts a menu item, accepts a YES/NO response, or skips to the next field of a display. If the numeric entry is the last of a display, ENTER steps to the next display.

COMMON 8Y8T~ 9600 DI8PLaY8 PPO~RAY diJplay Example Prog ~t~ Msg Temp CYCL tl7 Done 74 0C
Menu ~ UN-STORE-PRINT-HOME

Prog is either HOLD, CYCL, AUTO or METH
~## is the program t (1-150) or ??? if it is not stored yet Msg is either Done, Error, Abort-or blank Temp is the current ~ample temperature Menu are the avail~ble options R~NTI~E di-pl~y Example Action Temp Ramp to 94 0C 29 6C
Timer Prog/Cyc 10 00 Cycle 14 Action is either 'Hold at xx xC' or 'Ramp to xx xC' Temp is the current sample temperature Timer counts down the hold or ramp time or counts up a hold time of FOREVER~5 Prog/Cyc for a HOLD file is 'Prog xxx' for ~ CYCL or AUTO file is 'Cycle xx' - counts up MORE di-pl-y Example Setpt Tot Cyc Setpt t3 Tot Cyc 25 Timer Prog Hrs left 2 5 Proq 17 setpt i5 the current ~etpoint t ~1-9) - counts up Tot Cyc is the total t of cycles tl-99) in the current program Timer is the time left in the program in hrs - counts down Prog is the current program t (1-150) ~YPAD ~AU8E ~isplay Example Prog ~ Temp AUTO tl8 55 0C
PAUSE Timer PAUSE 9 45 Prog is either HOLD, CYCL, AUTO or METH
25 ### is the program t (1-150) or ??? if it is not stored yet Temp is the current sample temperature Timer is the configurable pause time - counts down TOP L~VEL ~8ER IN~ERFACF

Select Option 9600 ~N-CREATE-EDIT-UTIL
TOP LEVEL display Run Cre~te program Enter program #xx _ _ OLD-CYCL-AUTO-METH
RUN display CREATE display Edit Select function Enter program #xxx ~ IR-CONFIG-DIAG-DEL
EDIT display UTIL display Programs are created by selecting a program type in the CREATE display. The user is brought directly to the first display of the program to be edited. -5 Stored programs are retrieved by entering a number 1 to 150from the RUN, EDIT, or program displays. Entering a valid program number from the RUN display automatically begins the run. Entering a valid program number from the EDIT or program display brings the user to the first display of the 10 program to be edited.
Programs are edited by pressing STEP (move down a ~creen), BACK (move to the previous field) or ENTER (move to the next field).
Programs are run by selecting RUN the ~UN-STORE-PRINT-HOME
menu or by pressing the RUN key on the ~eypad. The user must first enter 2 parameters required for each run.
The OPTION ~ey toggles the tube 20 Tube type. MICRO type from MICRO (MicroAmp tube) Re~ct vol- 100uL to THIN (thin-walled GeneAmp tube). If the user configured a ~pecial tube, then the option of OTHER is ~dded. A different reaction volume may be entered.
These parameters are stored with this program. ENTER accepts these values.
If the user configured the Se~ect print mode runtime printer ON and he is OFF-CYCLE-SETPOINT running a cycle, auto or method ~ program, then the following ~ . . ~

printer choices are offered. the program is started. CYC~E prints a message only upon completion of a cycle. SETPOINT prints runtime data for every ~etpoint (ramp/hold time ~nd temps).

. . . ~

CA 022660l0 l999-04-Ol If the user configured the Select print mode runtime printer ON and he is OFF-ON running a hold program, then the _ following printer choices are offered.

If the heated cover i8 below Cover temp i6 YX-C 100~C, the following acreen is Run starts at 100-C displayed. If thc u~er ~- on this di~play when the heated cover reaches 100~C, the run automaticaIly begins. If the user hit STOP to return to the program d-ispl~y, tben the run must be manually re-~tarted.
15 Accepting HOME at the RUN-STORE-PRINT-~OME menu without saving a program displays the 6creen:
Prog txxx not ~tored Continue? YE~

~OLD ~xxx xx.xC
_ UN-STORE-PRINT-HOME
PROGRAM display The user can choose between an Hold at xx.~C inf nite ~oak or a time limited Hold FOREVER-xxx:xx hol .

The beeper will ~ound once a Beep while Hold? NQ second.

HOLD ~ROGRAM - Ru~tim- disp~ay-Hold at xx.xC xx.xC None xxx:xx Proq xx RUNTIME display MORE di~play HOLD ~xx xx.xC None PAUSE xx:xx KEYPAD PAUSE display PROGRAMMED PAUSE

80~D P~OGRA~ - ~uut$-- prlntout PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 10 xx:xx am Tube type:MICRO Reaction vol:lOOuL Start clock wit~in x.xC
of setpt HOLD program ~xxx HOLD Program: xx.xC xxx:xx Actual: xx.xC xxx:xx or HOLD Program: xx.xC FOREVER Actual: xx.xC xxx:xx HOLD program ~xxx - Run Complete Nov 14, 1990 xx:xx am CYC~ P~OGRU~

CYCL ~xxx -xx.xC
UN-STORE-PRINT-HOME
PROGRAM display The default is 3. This Temperature PCR determines the number of ~etpoints in this ~Gy~am. 1 to 9 setpoints ~re allowed.

The number of setpoints entered Setpt ~1 Ramp x~:xx above determines how many xx.xC Hold xx:xx ~etpoint edit displays will be offered. The user can enter a ramp and hold time for each ~etpoint. The hold timer will start when the sample temp gets within a user configurable temp of ~etpoint.
If the user does NOT want to Total cycles - ~x pause, then the next 3 displays Pa~se during run? NO are skipped. 1 to 99 cycles are allowed.

Entering a O for setpoint number Pause after setpt ~ also means the user does NOT
Beep while pause?YES want to pause therefore the next 2 displays are skipped.

The cycle number is limited to l~t pau~e ~t cycl Y~ the total number of cycles Pau~e every xx cycl8 entered ~bove.

The default pause time is set in Pause time x~:xx the user configuration.

CYCLE ~ROGRAM - Runtio- di-pl~y-R~mp to xx.xC xx.xC Sctpt ~x Tot Cyc xx xxx:xx Cycle xx ~rs left X.X Progxxx RUNTIME display (ramp) MORE di6play Rold at xx.xC xx.xC
xxx:xx Cycle xx RUNTIME display (hold) CYCL ~xxx xx.xC Setpt ~x xx.xC
PAUSE xx:xx PAUSE xx:xx Cycle xx REYPAD PAUSE display PROGRAMMED PAUSE

CYCLE PROGRAX - Ru~t$m- printout PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx:xx ~m 5 Tube type:MICRO Re~ction vol:lOOuL St~rt clock within x.xC
of ~tpt CYCL progr~m ~xxx Cycle ~xx Setpt ~x RAMP Program: xx.xC xx:xx Actual: xx.xC
10 XX:XX
HOLD Progr~m: xx.xC xx:xx Actual: xx.xC
xx:xx . (up to 9 setpoints) . .
(up to 99 cycl~s) CYCL program ~xxx - Run Complete Nov 14, 1990 xx:xx ~m CYCL program ~xxx - User Aborted Nov 14, 1990 xx:xx ~m (only 20 if ~borted) A~TO ~ROGRAM

AUTO ~xxx xx.xC
UN-STORE-PRINT-HOME
PROGRAM display The default is 3. This Te~perature PCR determines the number of ~etpoint~ in this progrAm. 1 to 9 setpoints~re allowed.

The number of ~etpoints entered Setpt #1 xx.xC above determines how many Hold for xx:xx 6etpoint ~dit displays will be offered. No ramp time is offered thus the instrument r~mps as fast a~ possible. The hold timer start when the sample temp gets within a user configurable temp of setpoint.
If the user wants to increment Setpt ~1 xx.xC or decrement the time and/or Change time/temm~YES temperature every cycle, then _ the following display is offered.

The OPT~ON key toggles the arrow xx.xC delta _ x.xC up (increment every cycle) or delta xx:xx down (decrement every cycle).
The max time allowed to decrement is limited to the setpoint hold time.
Up to 99 cycle6 are allowed.
Total cyc}e~ - X~

AVTO P~ W RA~ - Ru~t~- display-Hold at xx.xC xx.xC Setpt ~x Tot Cyc xx xxx:xx Cycle xx Hrs left X.X ~o~AX
RUNTIME display MORE display AUTO ~xxx xx.xC Non-PAUSR XX: XX
KEYPAD PAUSE displ~y PRO~JR~MMED PAUSE

a~o PROaRAX - Runt~ - ~r~ntout PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 5 Tube type:MICRO Reaction vol:lOOuL Start clock within x.xC
of setpt AUTO program ~xxx Cycle ~xx Setpt ~x RAMP Program: xx.xC xx:xx Actual: xx.xC
10 xx:xx HOLD Program: xx.xC xx:xx Actual: xx.xC
xx: xx (up to 9 ~etpoints) . (up to 99 cycles) AUTO program ~xxx - Run Complete Nov 12, ~990 xx:xx am AUTO progr~m ~xxx -.User Aborted Nov 12, 1990 xx:xx am ~only 20 if aborted) .

~TUOD PROGRAM

METH ~xxx xx.xC
UN-STORE-PRINT-HOME
PROGRAM display Up to 17 programs can be linked in a method. If the user tries Llnk progs: - - to enter a non-existant program ~, the me~sage ~Prog does not exist" i6 displayed. If the user tries to link another method, the message "Cannot link a method" is displayed.

M~T~OD PROaRAM - Runt~ - d1-P1aYJ
The RUNTIME, MORE and PAUSE displayc will be those of the program currently running. Two additional MORE displays are offered when the program running is linked in a method.

The number of the program ME~ ~xxx aaa-bbb- currently running will flash.
ccc-ddd-eee-fff-qgq-ADDITIONAL MORE display .

bhh-iii-~ j ~ -kkk-lll-mmm-nnn-ooo-ppp-qqq ~ OD PROaRAN - Ru~t~- pr~ntout PE Cetus GeneAmp PCR System 9600 Ver xx.x Nov 14, 1990 xx:xx am Tube type:MICRO Reaction vol:lOOuL Start clock within x.xC
20 of setpt MET~OD program ~xxx - preceeds all linked program data CA 022660l0 l999-04-Ol METHOD program ~xxx - Meth Complete - follows all linked progra data ~T~OD PROGRAX - Print Select option y ETHOD-PROGRAM DATA

METHOD prints the header of each program linked in the method.
PROGRAM DATA prints the header and contents of each program linked in the method.

8TOR~NG A PROGRAM
When STORE is selected from the RUN-~TORE-PRINT-HOME menu, the routine for ~toring a program is the ~ame for a file as well as a method. Protecting a program insures the user 5 that the program will not be overwritten or deleted without knowledge of the user number. Other users will be able to view, edit, run, and link the protected file in their methods but will not be able to alter the stored version.
xxx i6 the first available 10 store program number from 1 - 150.
Enter program ~ ~
The user has entered the ~ of a Progxxx is protected protected program. The correct Enter user ~xxxx user # must be entered in order ~ to overwrite this program.
.

The wrong user ~ was entered.
Progxxx is protected This display remains for 5 Wrong user number! 6econds before reverting to the previous one. The user is given 3 chances to enter the correct #.
If the user tries to overwrite a Progxxx is linked in program that is linked in a Methxxx Continue?YES method, the user ~s warned and _ given the option of continuing or not.
If the user tries to overwrite a Can't overwrite prog program that is linked in a Linked in method xxx method with another method, an error mes6age is given.

The u~er i~ given the chance to Store protect a program as well as Protect program? N ~ unprotect a previously protected program.

The user wants to protect the Store program and therefore mu6t enter Enter user ~xxx~ a u~er #.

Ready to store the program in an available 610t. The user # appears only if the program is protected.

Prog ~xxx User ~xxxx Ready OK to store? YES Prog txxx U~er ~xxxx ' OK to overwrite? YE ~ it an existi n g progra m. The user ~
appear s only if the progra m is protec ted.

~ILITY FVNCTION~

Select function IR-CONFIG-DIAG-DEL
UTIL display DI~ allow the user to view or print ~ directory of the stored program~ by either their program number, user number or program type. .
5 coNFIa allows the u~er to tailor the use of the instrument to their specific needs.
DIAG offers the user a means of diagnosing runtime problems and verifying the performance of the instrument.
~0 DEL allows the user to delete stored programs by program number, user number or program type.

VTIL - DIRECTORY

Directory PROG-TYPE-USER-PRINT

Dir-ctory by PROGr~ nu~b-r P.oy~ams will be listed in Directory numerical order starting at the Enter program ~ ~ given number. The STEP ~nd 8ACK
keys move through the directory displays. The beeper sounds at the beginning or end of the program list.
STOP returns the user to the HOLD ~124 above display.

Dir-ctory by progr~ TYP~
The program numbers will be Directory listed for the selected type of 15 HOLD-CYCL-~UTO-Mr.Th program.

CYCL ~15 Dir-ctory by V8~R uu~b-r All ~L G~ amB stored under the Directory given user number will be Enter user ~xxx~ listed.

M T~ ~lSO ~ser ~1234 20 Dir-ctory PR~NT

The user can get a hardcopy of Directory Print the directory listing in the ROG-TYPE-USER ~ame ~anner the directory is viewed ~bove.

VTIL - l;J8E:~ CONFIG~JRATION
The configuration file can be Configuration edited by ~ccepting EDIT from EDIT-PRINT the menu or by pressing the STEP
5 _ key. PRINT prints the contents of this file.
The user can set the system time Time: xx:xx and date.
Date: mm/dd/yy If the runtime printer is ON, 10 Runtime printer OFE the user will be prompted with Runtime beeper ON prlnter optlon as the start of each run. If the runtime beeper is ON, then a beep will sound at the end of each segment (after a ramp or hold portion of a sequence) while running a program.
This time represents the maximum Pause time-out limit amount of time a program can 20 xx.xx pause for before it is aborted.
- This pertains only to the keypad pause.
This time represent~ the number Allowed setpt error of degree~ the actual sample 25 x.x~C temp may vary from the ~etpoint before an error is flagged.

This setpoint is useful for Idle ~tate 6etpoint balancing the control cooling xYoc power which is always present.
30 _ The sample temp will be maintained at the idle ~tate aetpoint whenever the instrument is idle.
The clock which times the hold 35 Start clock within 6~ ,' ent of a running program can x.x~C of ~etpoint be configured to be triggered when it gets within this temperature of the ~ample temp.
The nominal value is 1.0~C.

If the user wishes to use a different type of tube other than the MicroAmp or Thin-walled GeneAmp tubes, they ~ust set this option to YES and enter ~t least 3 pairs of , reaction volume and tube time Special tube? NQ constant data. This curve will be used to extrapolate the correct Tau (tube time constant) for each run using this special tube depending on the reaction volume entered by the user at the start of a run.

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~TIL - ~8~R CONFIG~RATION ~cont) 3 ~ets of this screen will be Rxn vol~xxxuL ~~ x~ offered if the user ~ets Rxn vol-xxxuL '~ 5 ~Speci~l tube?" to YES.

V~I - DELETE

Delete PROGRAM-USER-ALL

D-l-t- by PROGRAM
All programs (files and methods) Delete can be deleted by number.
Enter program ~xxx _ A program cannot be deleted if Can't delete progxxx it linked in a method.
Linked in methodxxx!

The user has entered the t of a Progxxx is protected protected program. The correct Enter user ~xxxx user # must be entered in order lo r to delete this program.

The wrong user ~ was entered.
~ Gy~ is prot-cted This display remains for 5 Wrong user number! seconds before reverting to the previous one. The user is given 3 chances to enter the correct ~.
Ready to delete the program. The Prog ~xx ~s-r ~xxxx user ~ ~ppears only if the Delete proqram? YES program wa~ protected.

20 D-l-t- by V8ER
Programs can be deleted under a Delete given user number.
Enter user ~xxx~

If no ~ Gy~ ams exist with the Delete given user ~, the following 25 No progs with ~xxxx message is displayed.

, Programs cannot be deleted if Progs linked in meth they are linked in a method. ~he STEP to list progs STEP key will cycle through the list of linked programs.

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~TIL - DE~E~ ~co~t) The liet of the linked programs Can't delete ~o~ will show which method the Linked in methodxxx! p~Gy~am ie linked to.

This will delete ~ll the User ~xxxx programs under the given user Delete all progs?YE ~ that are not linked.

D-l-t- AL~
This will delete every Delete everY unprotected program that is not unprotected prog?YE ~ linked in a protected method.

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~ 8~R DIAGNO8TIC8 While running any diagnostic test, the STOP key always returns the user to the top level diagnostic ~creen and automatically increments the test number and name to the 5 next test. This facilitates manually cycling through the available diagnostics.
The user can enter the number of ~ Enter Diag Test ~l the diagnostic to run or can use ~EVIEW HlSTORY F~LE the STEP or BACK ~eys to cycle through the available tests.
Every time the STEP or BACK key is pressed, the test number is incremented or decremented and the associated test name is displayed. This feature eliminates the need for the user to memorize the number associated with each test.
REVIE~ EI8TORY PIS~
The history file is a circular Enter Diag Test ~ buffer in battery RAM which can R~VIEW HISTORY EILE store up to 500 records of the latest run. When the buffer is full, the oldest entries will be overwritten. The buffer will automatically be cleared before -- a program is executed.
The history file header displays HISTORY nnn recs the current number of records in ALL-STAT-ERRORS-PRNT the file ('nnn').
aLL views all the records 8TAT views only the status - records ~~~OPQ views only the records with error messages ~RNT prints all or part of the history file The two types of records are l) status records which give information about the program and 2) data records which give 40 information abount each hold and ramp segment in a program.
A Hold program is treated as one hold segment and the data record will be stored when the file ends.
Since there could be hundreds of entries (50 cycles X 6 setpoints ~- 350 entries), fast, bi-directional movement 45 through the file i8 required. Note that most PCR programs will be 3 or 6 setpoint~ and 40 cycles or less. The entries will normally be reviewed in reverse order, thus the first record eeen will be the last record written.
If the user has chosen a type of record to view, STEP or BACK will move down or up the buffer by one entry of the chosen type. By preceding STEP or BACX with a number, the 5 second line is replaced with "Skip ~XXX entriesn. The user enters a number and pre6ses ENTER to accept the value and that number of entries is skipped going forward (STEP) or backward (BACK).
By preceding STEP or BACX with the RUN key, the user can l0 quickly move to the largest record ~ (the newest record) or record ~l (the oldest record) of the cho~en type.
STOP terminates the review mode and di~plays the file header.

STAT~8 pr~pn 'ffff' is either HOLD, CYCL or ffff ~xxx/mmm nnn AUTO
~essage 'xxx' ~s the program number '/mmm' is the method number for a linked program, else blank 'nnn' is the record number 'message' is one of the following:
8t~tu~ g--10 Tube Type: xxxxx Type of sample tube used in the run Reaction vol: xxxuL Reaction volume used in the run Clk starts w/in x.xC The hold clock starts within this temp of setpoint Start xx/xx/xx xx:xx Time and date of the start of the run 15 End xx/xx/xx xx:xx Time and date of the end of the run Meth Complete All programs linked in the method are complete Pause xx:xx at xx.xC The program paused for this time at this temp 20 F~t~l ~tatu- m-~ag--Sensor Error A ~ensor had a bad reading lO
times in a row Power fail xxx.x hrs The power was off for this amount of time 25 User Abort - The user pressed the STOP key during the run Pause Timeout xx:xx The keypad pause has reached its configurable time limit.
Fatal Setpoint Error Is the requirement to abort a program if the setpoint is not reached within a calculated amount of time. A lO X
l0 lookup table of starting ramp temperature (0~C - l00~C in l0~C
increments) vs. ending ramp temperature (same axis labeling) will hold the average time the TC2 ~hould take to ramp up or down ~ny given ~mount of degrees. The file will be aborted if the setpoint is not reached in the amount of time calculated as follows:
programmed ramp time + (2 ~ lookup table value) +
l0 minutes DATa p~o~n 'f' is either ~OLD, CYCL or aUTO
'xxx' is the program number .

'/~mm' is the method number for f#xxxJmmm ddd.dC nnn ~ linked program else blank Cycyy Setpt z mmm:ss 'ddd.d' i8 the ending setpoint 'nnn' is the record number 'yy' is the cycle number 'z' is the setpoint number ~mmm: SS ~ i8 the setpoint time The cycle ~nd ~etpoint number fields will be omitted for Hold progr~m.~

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DATA FM 0~ pFcopn 'ddd.d' is tbe ending setpoint me6~age ddd.dC nnn temp Cycyy Setpt z mmm.ss 'nnn' is the record number 'yy' i~ the cycle number 'z' is the setpoint number 'mmm:ss' is the setpoint time 'message' indicates a non-fatal error as follows:
10 Non-f-t-1 ~rror n--sag--Setp Error The setpoint was not reached in the calculated time:
programmed ramp time + (2 * lookup table value).
15 Prog Error An Auto program auto increment/decrement of the ~etpoint temp or time caused the hold time to go negative or the temp to go out of the range 0.1~C to 100~C.
~ Temp Error At the end of the segment, the setpoint temp has drifted +/- a user configurable amount.
For the Hold program, the cycle and setpoint fields will be omitted.

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PRINT~NG ~ H~8TORY r~L~
Access to the history file print routines is through the history file header menu. The OPTION key cycles the cursor through the options:

RISTORY nnn recs ALL-STAT-ERRORS- ~

5 Pressing the ENTER key when the cursor i~ positioned under PRNT displays the print screen:

Print History ~ LL-STAT-ERRORS

ALL prints all the records in the file STAT prints only the status records ERRORS prints only the records with error messages 10 When one of print options is selected, the following screen is displayed:

Print History Print from proq ~xx The first (most recent) program number will be the default program. The user can change the program number from which to begin printing. While printing, the following screen i~
15 displayed:

Print Hi~tory ...printing At the end of printing, the Print History menu is again displayed.

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~ATER T~8T

Enter Diag Te8t ~Z
HEATER TEST

The heater test calculates the heat rate of the ~ample block as its temperature rises from 35~C to 65~C The following ~creen is displayed a~ it forces the block temperature to Heater Test Blk-XX X
going to 35C

When the temperature ~tabilizes, all heaters ar- ~Ul ed on full power The display now reads ~going to 65C" and the block temperature i6 monitored for ?0 second6 a'ter it passes 50~C After 20 seconds, a pa6~ or fail me~sage is 10 displayed Heater Test PASSES

C~ILL~R TE8T

Enter Diag Te~t CHILLER TEST

The chiller test calculates the ~ool r~te of the eample block as its temperature drops from 35-C to 15~C. The following ecreen is displayed a~ it forces the block 5 te~perature to 35~C.
-Chillr Test Blk-XX.X
going to 35C...

When the temperature ~tabilizes, the chiller is on. The di~play now reads "going to 15C" and the block temperature is monitored for 20 6econds after it passes 25~C. -After 20 seconds, a pass or fail message i~ displayed.

C~iller test PASSES
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Claims (18)

CLAIMS:

1. A two-piece plastic holder for loosely holding a plurality of microliter sample tubes of a preselected design, each having a cylindrically shaped upper section open at its top end and a closed, tapered lower section extending downwardly therefrom, each tube being of circular cross section and having a circumferential shoulder extending outwardly from said upper section at a position on said upper section spaced from the open end thereof, comprising:
a. a one-piece tray member, comprising:
i. a flat, horizontal plate section containing holes in a rectangular array compatible with industry standard microliter plate format, said holes being slightly larger than the outside diameter of the upper sections of said tubes but smaller than the outside diameter of said shoulder;
ii. a first vertical tray sidewall section completely around said plate extending upwardly to a height greater than the height of a tube resting in one of said holes;
iii. a second vertical tray sidewall section around said plate extending downwardly approximately to the bottom of the upper section of a tube resting in one of said holes; and b. a one-piece retainer releasably engageable inside said tray over any sample tubes resting in said tray, comprising:
i. a flat, horizontal plate section containing holes in a rectangular array compatible with industry standard microliter plate format, said holes being slightly larger than the outside diameter of the upper sections of said tubes but smaller than the outside diameter of said shoulder; and ii. a vertical retainer sidewall section around said retainer plate section extending upwardly from said plate, wherein when said retainer is engaged inside said tray, the retainer plate section lies slightly above the shoulder of a tube resting in said tray and the first tray sidewall section is about as high as said retainer sidewall section, whereby tubes resting in said tray are retained loosely both vertically and laterally.

2. The holder according to claim 1, wherein the holes in said tray member are countersunk and wherein the underside of the shoulders of said tubes are correspondingly beveled.

3. The holder according to claim 2, wherein the holes in the tray plate section and in the retainer plate section are larger in diameter than said tubes by about 0.7 mm.

4. The holder according to claim 1, 2 or 3, wherein said tray member further comprises a plurality of support ribs extending along the underside of the tray plate member between rows of holes, said ribs extending downwardly to the same extent as said second vertical tray sidewall section.

5. The holder according to any one of claims 1 to 4, wherein said tray member further comprises a skirt section extending at least partially around said tray plate section and depending vertically from that section, said skirt section being adapted to fit into a guard band groove in a thermocycler sample block.

6. The holder according to any one of claims 1 to 5, wherein said tray plate section has at least two openings provided therein and said retainer plate section has an identical number of vertical tabs, downwardly extending from said retainer plate, such that said tabs project through said openings and releasably engage the tray when said retainer is assembled with said tray.

7. The holder according to claim 6, wherein said tabs are disposed so as to form part of a skirt section extending downwardly at least partially around said tray plate section and wherein said tabs are adapted to fit into a guard band groove in a thermocycler sample block.

8. The holder according to claim 7, wherein said openings and said tabs are positioned such that said retainer and said tray are capable of only one orientation relative to one another when said openings and said tabs are engaged.

9. The holder according to claim 6, wherein said tabs are deflectable in a sidewise direction in order to come into alignment with said openings.

10. The holder according to any one of claims 1to 9, further comprising up to 96 microliter sample tubes in said holder.

11. The holder according to claim 10, further comprising up to 96 deformable caps on said tubes for forming gas-tight seals thereon.

12. The holder according to claim 11, wherein each said cap has a downwardly depending cylindrical flange for forming a gas-tight seal with each said tube and a circumferential shoulder extending outwardly from said flange which prevents said flange from being seated on said tube below a predetermined point.

13. The holder according to claim 12, wherein the outer circumference of said downwardly depending flange fits snugly to form a gas-tight seal with the inner circumference of said tube.

14. The holder according to claim 11, wherein groups of 12 of said caps are linked together to form a single strand of caps which are suitably spaced so as to form gas-tight seals with up to 12 of said tubes.

15. The holder according to claim 1, further comprising a plastic base having 96 wells arranged in an 8-by-12 rectangular array, said wells being dimensioned to snugly accept the lower sections of up to 96 said sample tubes, said base being assemblable with said tray, said retainer and 96 of said tubes to form a microliter plate having the footprint of an industry standard microliter plate.

16. The holder according to claim 11, wherein said caps project above said first vertical tray sidewall section but are downwardly deformable to the height of said section.

17. The holder according to claim 16, wherein said caps are deformable by heat and vertically downward force.

18. The holder according to claim 16, wherein said caps are resiliently deformable.