CA1299772C

CA1299772C - Scanning fluorescent detection system

Info

Publication number: CA1299772C
Application number: CA000564536A
Authority: CA
Inventors: Charles William Robertson Jr.; Rudy Johan Dam; James Merrill Prober
Original assignee: EI Du Pont de Nemours and Co
Current assignee: PerkinElmer Health Sciences Inc
Priority date: 1987-06-12
Filing date: 1988-04-19
Publication date: 1992-04-28
Anticipated expiration: 2009-04-28
Also published as: AU1762088A; IL86695A0; DE3851910D1; JPS63317770A; AU601048B2; IL86695A; US4833332A; EP0294996A3; EP0294996B1; DE3851910T2; EP0294996A2

Abstract

Title IP-0678 SCANNING FLUORESCENT DETECTION SYSTEM
ABSTRACT
A system for detecting the radiant energy emitted from different closely spaced species includes two detectors each having a large entrance angle for receiving the radiant energy, and wavelength selective filters between the detector and species, the transmission vs. wavelength characteristics being complementary, and means for ratioing functions of the detector outputs, the ratio being indicative of the identity of the species.
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Description

- ;-Title IP-067 SCANNING FLU~ESCENT DETECTION 5YSTEM
ield of the. ~nvention Thi~ invent~on selates to a scanning ~luorescen~ detection sy~em and, ~ore par~icularly, to apparatu~ fiuitable for u6e with a fluorescen~e-ba~ed DNA ~equencer. Th~s ~y~tem i~
~apable of di~tin~u~6hing among 6imilar fluoeophores with eelat~vely low lev018 O~ eml~sion. ~ unique arrangsmene of ~ ~ileer and riber optic faceplate enable~ the ~y6ee~ to ~o~itor signals ro~ relaeively ~ large dete~tion areas ~ont~ini~g ~ultiple 6ample ~egions vhile ~till tetaiaing the re~uired opt~cal ~haraeteristic~ of the comb~ned ~1ters.
15Backqround of ehe Inventio~
DNA geq~lencing, i.e., det~inin~ the ~equenGe or order of the ~ucleo~ides or ba~es compri~i~g the DNA, i~ o~e of the corner6to~e analytical technlque6 of ~oder~ aoleculae biology.
The de~elop~ent of e~liable method6 ~or sequeneing ha~ led to great advance~ in ~he understanding of the organization of genetic information and ha~ ~ade pos6ible the ~anipulat~on o~ qenetic material. i.e., yenetic engineering.
There are currently two geneeal methods for 6equencing DNA: the Maxam-Gilbert chemical degradation method [A. M. Maxam et al., ~eth. in Enzym. 65 499-599 (1980)1 and the Sanger dideoxy chain ter~;nation method ~F. Sange.r, et al., Proc.
Nat. Acad. Sci. USA 7~ 546~-5467 (1977)J. A common feature of the6e two technigue~ is the geneEation of four group6 of labeled DNA ~ragment~, aach group ~aving a family of labeled DNA fragments with each amily coneaining fragme~ts having differing number~
o~ nucleotide6. ~he population of fragment~ within t~e6e group~ all end with one of the four nucleotide6 or base6 compri6ing the DNA. Both technique~ also utilize a radioactive i60tope, such a6 3~P or 35S. as the means for labeling ~he ~raqment~. The primary difference be~ween the technique6 i8 in the way the fragment6 are prepared.
l~ both ~ethod~, ba~e ~eguence information which generally sannot be directly deeermined by physical ~ethod6 mu6t be converted into chain-leQgth information whic~ can be de~er~ined. This deter~ination ca~ be acco~pli6hed through electrophoretic 6eparation. -Unde~ de~aturi~g condition~ (hiqh temperature, urea pre~ent, etc.) shor~ DU~ ra~ment6 ~igrate through the electrophoresi~ medium as tiff ~ods. If a gel i6 employed for the eleoerophoresi6, the DNA fraqment6 will be 80rted by 6i2e and re~ult in a DNA seguence deteemination wit~ ~ingle-base resolution up to ~e~eral hundred ba6e~.
The Sanger and ~axam-Gilbert ~ethods for DNA
6equen~ing are c4nceptually elegant and efficaciou~
but they are operationally difficult, time-consuming, and often inaccurate. Many of ~he problem6 6tem from the fact that the single radioi60topic repoeter cannot di~tingui6h between ba6e6. The use of a single reporter to analyze the sequence of four ba6e6 lend6 con6iderable complexity to the overall proce66. To determine a full 6eguence, the ~our 6et6 of feagment6 produced by either ~axam-~ilbert or Sanger methodoloqy are sub3ected to electrophore~is in four parallel lane~. Thi6 re6ults in the frag~ent~ being 6pacially re~olved along the length S of the gel according ~o ~heir 6i2e. The pattern o~
labeled fragment~ ypically read by autoradiography which show~ a continuum o~ bands distributed between ~our lanes often referred to as a sequencing ladder. The ladder i6 read by vi~ually ob~erving the film and determining the lane in which the nex~ band occur~ for each 6tep on the ladder.
~hermally ~nduced distortion6 in ba~e ~obili~y in ~he gel lthi6 u6ually appear6 a~ a "6mile effect" across the gel~ can lea~ to dif~icultie6 in ~omparing the four lanes. The~e distortion~ often li~it the number of ba~e~ that can be read on a 6ingle gel.
Proble~s rela~ing to use of a 6inqle radioisotopic reporter revolve around i~s lack of sen~itivity and the ti~e required to e~aluate a ~ample. The long time~ required for autoradiographic i~aging along with the nece~ity o~ u~ing four parallel la~es force one i~to a U6nap6hotu ~ode of vi6ualization. Si~ce one ~eed~ ~imul~aneou6 ~patial resolution of a large nu~ber of band~ one i~ ~orced to u6e large gel6. This re~ult6 in additional problems. Large gels are difficult to handle and are 610w to run, adding even ~ore ~ime to the ov2rall proces~ .
once the expo6ed image of the gel pattern i~
obtained, ~here is a problem of vi6ual interpretation. Conver6ion oP a ~equencing ladder into a ba6e ~equence i6 a time-inten6ive, error prone proces~ requiring the full attention of a highly skilled operator. Some mechanical aids do exist but the proce~6 of interpreting a sequence gel i6 6till , . ;. ~

~2~77~

pain~taking and slow. Finally, the ~se o ~adioactive ~aterials ha6 healeh ri~k~ a~30ciaeed with continued expofiure over ex~ended periods.
Appropriate u~e of shielding and di~posal proeedure6 imposes some con~rol of expo6ure level6, but elimination of i~otope u~e would be highly desirable.
~ o solve these problems, effort~ are underway to replace autoradiography wi~h 60me alternate, non-~adioi~otopic reportertdetection ~y6tem usinq fluorescence. DNA fragment~ labeled with one or ~ore ~luore~cent tags (fluorescent dyes) and excited with an appropriate light ~ource give ~haraceeristic emi6sions ~rom the tags which identify the frag~ent6.
lS The u~e of a fluore6cent tag a6 opposed to a radioi60topic label allows one to speci~y a DNA
frag~ent detectio~ ~ystem thae res2ond~ to the optical parameter~ characterizlng tag fluore6ce~ce.
For e~ample, the use of fvur ~if~eeent ~luore6cent tag6, diLti~guishable on the basis of ~ome em~s6ion charac~eristic (e.g., 6pectral di6tribution, life-time. polarization), allow6 one to uniquely link a q~en t~g with the sequenci~g fragment6 as~ociated with a given base. Once 6uch a linkaqe i6 establi6hed, one zan then combine and re601ve the fragment6 f~om a single 6ample and make the base a6signmene directly on the basi6 of ehe chosen emis6ion characteristic. When electrophore6i6 i~
chosen as a separation means, for exa~pleO a ~ingle 6ample containing DNA fragments with ba6e-specific fluorescent tags can be separated in a 6ingle gel lane.
The "real-time" nature of fluore6cence detection allow6 one eieher to scan in the electrophore~is direction a gel containing spatially . ..

re~olved bands (ra~olution in ~pace) or to monitor at a ~ingle point on the gel and detect band6 o~
~eparated fraqmen~s as they pa6~ in sequence through the detection xone (resolution in time). Large gel6 are no~ necessarily required to di6criminate between the ~ragmen~ when time re~olution is 6elected.
~urthermore, a "real-time", 6ingle lane de~ection moda i~ very amenable to fully automated ba~e a6~ignment and data tran6fer.
~ known "real time" fluore6cence-based ~NA
seguencing 6ystem developed by the California In~titute o~ Technoloyy ~ disclo6ed i~ at lea~ one publi6hed pate~t application and at lea~t ~wo journal article6: L. ~. S~ith, We~t German Pat. Appl. DE
15 3446635 Al 11985); L. ~. S~ith et al., Nu~leic Acid8 aesearch. 13 2399-Z412 (1985) and L. M. S~ith et al., ~ature, 321: 67~-~79 (19~6). ~hi~ sy~eem employ6 our ~et~ of DNA ~equencing fragment6, each labeled with one o~ four specified fluore6~ent dye6.
Unfortunately, the fluore~cence (emi66ion) ~axima are 6pr~ad over a large wavelength tange (approximately 100 nm) to facllitate discrimination amonq the ~our dyes, but, the absorption (excitation) ~axima for ~he dye6 are comparably 6pread. Thi6 make6 it difficult to efficiently excite all four dye6 with a ~ingle ~onochromatic source and adequately detec~ the re6ulting emi~6ions.
It would be preferable to u6e dye6 with clo6ely ~paced ab60rption (and corre6ponding emis6ion~ 6pectra, selected to enhance the excitation efficiency. ~ut 6uch elo6ely 6paced 6pectra cau6e other difficultie~. Recall that a real time detection 6y6tem for DNA sequencing mu~t be able to distingui~h between four different dye emi66ion ~pectra in order to identify the individual labeled i: .. . . -~29g~

fragment~. The~e emi6fiions are typically of relatively low inten6ity. The detection 8y6tem must haYe a high degree of 6electivity and 6en~iti~ity (bette~ than lo (-16) mole~ DNA per band), and a mean~ to minimize ~tray light and backgeound noi~e.
in order to meet desired performance characteri~tic~. The ~y~tem mu~t al60 ~e able to monitor the detection area frequently enough to avoid missing any fragment6 Shat ~ay migrate pa~t Che detection window between scan~.
In order to effectiYely utilize an electrophoresi6 gel. a typi~al D~A ~equen~ing experi~e~t ln~olves running ~ultiple sa~ples si~ultaneously in pa~allel lanes of ~ ~lab gel.
There~ore. a~ excitation/detectio~ 8y8tem ~118t al~o be able to ~onitor ea~h lane of ~uch a gel ~t es6entially the ~ame tl~e. A ~yste~ ~ust be eapable o ~oni~oring a detection zone whi~h æpa~s the ~a~ority of the usable ~el ~idth. ~ypi~al ~eque~cing gel6 have lane~ ehat are ~-5 mm wide ~ith 1-2 ~m spaciag betwee~ lane6. Therefore, ~n order to ~onitor a lO lane gel, a de~ection fiy~tem mu6t excite and de ecC emi~ions ~rom a region typi~ally a6 wide as 70 mm.
Anot~er ~luore6~ence detection ~y6tem developed for similar application~, is disclo~ed in a Canadian Patent Application S.N. 540,947 filed June 30, 1987 by Prober et al. Thi~ application di~clo~e6 a 6y6tem for detecCing the pre6ence of Lluore6cent energy from dif~erent specie~, typically dye-labeled DNA, following 6eparation in time and/or 6pace, and identifying the specie~. A set of four label6 are ~hosen ~uch thaC all four are ef1ciently 2x~1ted by a single source, yet have 2mis~ion ~pectra that are ~imilar but di6tlngui6hable in wavele~gth. Since ,a ~d~Yw~

dif~erential perturbations in electrophoreti~
mobility of the attached DNA fragments a~e small, any disturbance to this behavior is minimized by using four tags that have similar molecular weight~, 6hape and charge.
The scheme oE Pro~er et al. provide~ for ~d~lating and ratioing the signals corresponding to the fluorescent ener~ie& in two different wavelength range6 to obtain a resultan~ 6ignal that dete~mines ~he identity of the species. A ~ichroic filter, with a tran6~issi~n/reflection characteri6tic that varies as a func~ion of wavelengt~, or two filter6 with passbands Shat vary as a function of wavelength, effect the modulation. Two detector~ are positioned respectively to receive the transmitted and reflected emis6ions and generate fir~t and 6econd ~ignal~
corre~ponding to the intensities of each. Preferably ~he dichroic filter characteristic has a relatively sharp transition from transmission to reflection 2~ which occurs near the center of the species emission spectra.
This 6ySeem overcomes many of the problems o~ Smith et al. and ha~ the ability to dis~ingui6h in real time between relativ~ly 6mall wavelength difference6 in emission ~pectra, while maintaining a relatively high degree of 6en~itivity. Further, the 6y~tem deliver6 a high portion of the u6able light onto the two photomet~ic detectors ~o maintain continuous monitoring of the gel containing the fluorescent specie6.
Both o~ the system6 described above operate a fixed light beam and fixed detectors which together can monitor only a single point within the monitoring region. In order to monitor more than one spacial position (lane or lane position) of a gel, either the light beam muse be scanned while providing a means to detect the emissions from the dyes, or the gel must ~g~772 be physically ~hifted while holding the beam fixed.
The la~ter of the ~wo al~ernative~, moving the gel.
i8 not alway6 practical 6ince a large electrophore6is gel along with it~ a6sociated buffer re6ervoie~ are phy6ically cumber60me. ~he oth*r alternative, moving the beam while ehe gel i~ ~tationary, ha6 ~t~ own problem~ since the detector~ mu6t ramain clo~ely ~oupled to ~ources of emis~ion ~o preven~ ~he entry of ~tcay ligh~ and maximize eollec~ion of the emi~ted 0 light.
one ~ethod of accomplishing thi~ ta~k i~ ~o ~hysically ~ove either of the two previou61y di6cussed detection ~y8tem6 and thei~ a660ciated optics and light beam 80 that several lanes i~ the 5 gel ace effectiYely scanned. Thi8 type of ~y~tem ha~
the di~adva~tage of being mechanically ~o~ple~ while i~trodueing addit~cnal noi~e into the ~ystem.
~eliability and the high Gosts a~sociated with thi6 type o~ ~y6tem would also be a concern.
A~other kno~n "scanning" detection system i~
ai6~ussed in U.S. Patent ~,764,512 issued to Green et al. Thi~ ~y~tem disclo6e6 a laser ~eanning electrophor~ic in~trument and 6ystem for determining the electroki~etic or zeta pote~tial of disp~r~ed 5 particles in an aqueou6 ~olution. Thi~ 6ystem utilize6 a qalvano~eter ~irror to ~can a laser light beam a~ro66 an elect~ophoresis ~ediu~. ~he 6y6tem i~
not capable of detecting ~ultiple ~a~ple6 moving perpendicular to the 6canninq motion o the ~eam.
Another ~canning system iB disclo~ed in U.S.
Patent 4,162,405, Chance et al. which de6cribe6 an apparatus ~or mea6uriny the heterogeneity o~ oxygen delivery ~o perfused and in ~itu organ6. A la6er i8 ~ployed a~ a flying 6pot 6canning excitation source 5 and u6es two photodetector~ to monitoe the emi66ion ... . . . .

~2~ 72 6iqnal and exci~ation wavelen~th light. Although an x~y scanner i6 u6ed to ~ove the la~er beam over the ~ample area~ the total scanned area i8 only I sm by 1 cm. ~A~ mentioned earlier for a multiple sample DNA
~equencer, a 6ample area 7 ti~e~ wider i~ needed).
To implement the scheme6 of Chance et al.
for a larye area. the detector~ mu~t be either larger in ~ize. or loca~ed Purther from the 6ample thus diminishing the collectio~ efficiency. Furthermore, the detection of elo~ely 6paced emi86ion 6pectra oP
relatively low light iaten6ities i~ the pre6e~ce of a ~uch ~o e ~nten~e excitatio~ ~ource requir~ the ~elective tra~s~is~io~ properties offered by inter~erence fil~er6. In order to ~onitor a relati~ely large ~pacial area, both large detector~
and large filter6 ~u~t be u6ed. Unortunately~ large ~nterference filter6 ~hat collect light e~en a large ~olid angle are subject to ~rans~i~6ion propertie6 which vary with the angle of.incidence of the light.
Thus. ~hen placed clo~e to the emi~6ion souree, light impi~ging on the filter with an angle of ~ncidence qreates than about 2Z degree~ c~n experience signifi~antly le~s eeJec~ion of the excitatio~ light than light at normal lncidence. Consequently, if the filter subtends a relatively large 601id angle with re~pect to ~he source o emi6~ion, the overall excitatio~ wavelength re~eceion properties of the - filter will be compromised due to leakage of excitation light entering at the higher angles of incidence.

Su~mary of the Inyention Many o~ the above noted problem6 of ~he prior art radiant energy detecting Ey6tem6 aee overco~e by thi6 invention which ha~ particul~r ~L2~!72 application to a DNA sequencing sy~tem. Thi6 invention find6 use in a sy6tem for detecting the presence of radiant energy from different 6pecie6, typically dye-labeled DNA, following ~eparation in time and~or space, and identiying the ~pecie6, the 8y6te~ having first dete~tion means respOnBiVe to the radiant energy emitted by ~he 6pecies or geneeating a fir t ~ignal thae varies in amplitude in a fir~t ~ense a~ a function of the nature of the ~pecie~, 6econd detection means respon~ive ~o the radiant energy foc ~enerating a 6econd 6ignal that varie6 in ampli~ude in a second ~ense differe~t than the fir6t ~en~e as a functio~ of the nature o the $pecie6, and third ~ean~ re~ponsi~e to the first and ~econd lS 6iqnal6 for obtaini~g a th;rd 6ignal corresponding to the ratio of function~ of the fir6t and second fiignal6, the amplitude o the third 6ignal being indicative o~ ~he identity of each of the 6pecie~.
The invention i6 an i~provemene of su~h system wherein ~he fir6t and second mean6 each include: a detector having a large 601id entranee angle positioned adjacent to the 6pecie~ to re~eive ~adiane energy emitted from the specie6, and a wavelength 6elective ~ltec ~eans pO6~ tioned between each re~pective de~ector and the ~pecies. each wavelength filter ~ean~ having tran6mi6sion v6.
wavelength characteeistics that are comple~entary, and wherein one of the ~irst and secvnd detection ~ean6 include6 a transmis6ion filter means for rejecting radiant energy incident on a detector at an angle greater than a predetermined value.
Preferably, the 6pecie6 are excited by a beam of r~diant energy from a la6er and the sys~em include~ means to 6eparate ~olecule6 (typically fragments of DNA) labeled with emitting 6pecie~ o~

. ..;

materials. The detection means are po~itioned on opposite sides of the region propagating the laser beam of energy in which beam i8 swept across the BeparatiOn meanfi ~o exci~e the species in ~equence.
5 The wavelangth selective filters have a transition in their transmis6ion v8. wavelength characteri~tic6 centeLed at about the middle of the specie~' radiant eneegy 6pect~a. The tran6mi6sion filter has an extra ~ural ab60rber a~ong plural optical fibers positioned ~o have parallel generatrices t~ansver~e to the f ir6 and second detectorL.
This sy6tem i~ optically efficient and doe~
not ~eguire the use of len~es or other collection optics. It i6 capable of a~d doe~, by the use of detectors having a ~ide entran~e angleO view la ge area~ capable of accommodating plural electrophoresis lanes. These plural lan~ are sequentially and repeti~ively scanned. Becau6e of these efPiciencies, ; ~he 6yste~ ca~ operate u6ing low levels of e~itted radiant energy. ~he only ~oving part required in the ~ystem i8 a~ oeti~al element which ePfects the laser gsanning. The use of th~ t~an6~ission filters and associated ~tra ~ural ~bsorber6 ~ubstantially reduce extraneous light l~pi~gi~g on the detector. The 6yste~ i8 capable Oe detecting and distinguishiQg the radiant energy emitted fro~ plural ~ources that emit energy at diff erent bu~ closely spaced wavelengths.

Blief Description of the Drawin~s The invention may be more fully under6tood from the following detailed descrip~ion thereof taken in connection with accompanying drawings which form a part of thi6 application and in which:
Figure 1 is an i60metric view of the electrophore6i6 gel slab showing plural sample wells and lanes;

, .

1~
Figura 2 iB a partial diagrammatic layout of a system con~truceed i~ accordance with thi6 invention ~or detecting ~he pres0nc0 o~ ~adiant energy from different source~ that each emit energy at dif~erent but ~106ely ~paoed wavelenyth~;
Figure 3 ;8 a ~ide elevation view o~ the detec~Dr~ er arrangemens for detecting the pre~ence o~ radiant energy;
Figure 4 i~ a graph depicting ~he complementary filter tran6mi~ion ~haracteri~tics a~
a function o~ waveleng~h;
Figure~ 5A, 5B, and 5C are f low diagram~
des~ribi~g ~he routine6 and ~ubroutine~ u6ed to obtai~ sequence of DNA ~ragments u6ing the 6y6tem6 of this invention.

Detailed De~criPtion of the Prefer~ed Embodiment The radiation from very clo~ely 0paced e~is~ion bands ~ay be dete~ted u6i~g the 8y6tem of 20 thi5 inve~eion. These ~losely 6pac~d emi6sion~ are produced fro~ ~re~elected ~pecie6 which typically a~t a~ reporters a~d are irrever6ibly bound to ~aterial6 that ace to be a~aly2ed. Ac~eptable ~eporter6 are gener~lly one or more spe~iQ6 cho~en Por their ability to emit radiation over a narro~ ranga of ~avele~gth6, typically between a 50 and 100 nm range, preferably over a 20 to 50 nm range. Preferably, the peak maxima 6hould be 6paced no clo6er than 2 nm.
One reporter spe~ies may be capable of emitting energy at more than o~e wavelength, depending upon the manner of attachment to the material6 of intere~t and the condition~ of analy6is in the 6y6tem.
However, individual reporter6 with unique e~i66ion chara~teri6ti~6 in the 6y6tem are more ~onventionally cho6en to emit radiation in the wavelenqth range to be detected. Since the prefeered for~ of the invention i6 directed to detecting reporter-labeled DNA ~equencing fragments, it will be de~cribsd in tha~ con~ext. It iB to be undar6tood, however, that the i~ven~ion ~ay be u6ed to detect any light emitting labelled ~ample6 and i~ partieularly advantageous where the emi6~ion radiation has clo6ely ~paced wavelength6. The invention may be u~ed ~o detect, for example, fluor~çen6e, chemilumine 6 cence, lo and the like. Thus dye labelled DNA 6equencing fragment~ are pas~ed through an elec~rophore~
apparatu6 for æeparation. For thi~ eurpo e, a6 i6 illustcated in Fiqures 1 and 2, the electrophore6i6 ~ay be carried out by a 6uitable ~lab 10 arrangement typically having a thicknes6 of about 0.3 ~n and about 40 centimeter6 long and 15 ~entimeter~ wide.
Other sizeg ~ay be u~ed ~s appropriate. Thi6 slab 10 ha~ a ~uitable gel 11. typically 6% polyacrylamide;
sandwiched betwee~ ~la~ or low fluore~ci~g plastic l~UppOltB 12 .
The 61ab 10 is typically placed in an upright po6ition in a holder with the upper ~nd of the slab 10 extending through and into a~ upper coneainer 16 holding a bu~fer 24 and downwardly into a 6econd container 14 al60 holding a buffer 18. ~he buffer solution could be any guitable buffer such a6 that obtained fro~ a 601ution con6i6ting of .lM tri~
.lM boric acid, and .05M Na2 EDTA, with a final pH
o~ approximately ~.3. In thi6 manner, the buffer contact6 the gel at either end of the 61ab in order eo make electrical contact therewith.
With thi6 arrangemen~. a sample containing reporter dye-labeled DNA fragment6 can be pipetted into cavitie6 15 that are created at the top of the gel 11 and define 6eparation lanes. The re~2rvo~r container~ 14 an~ 16 are filled with buffer solutionfi 18 and 24. An electrical circuit is then completed through ~he terminal~ Z0 in re~ervoir container6 14 and 16. A ~ui~able electrical field (~ypically 50 volt6~cm) i6 needed ~o obtain ~eparations for gel~ of thi~ particular length and thicknes~. ~he po6itive elec~rode i loca~ed at the lower end of the 61ab to cau~e the DNA fragments to migra~e downwardly. Under thes0 condi~ions, a~ the fragment~ migrate through the gel t~ey are separated 6patially into bands tnot ~hown).
The~e band6 are detected by the ~y6tem and apparatus of thi~ inventio~ a~ they ~igrate dow~wardly ~ a detect~on zone 19 located aear the 15 botto~ of the $1ab 10. In ehi6 zone 19, the DNA
fragments are ~rradiated by a la~er beam 32 o~
appropriate excitation wavele~gth and the ~ifferen~
repor~er~ attached to the ~everal fragment~ emit detectable radiatio~. Si~ce-the reporters ~nd their attach~ent to the DNA frag~ent~ are not thé ~ubject of thi6 inve~tion, ~uch ~ill ~o~ be deficribed i~
detail. ~owever. an example of app~opriate reporter6 ~s described in the copendi~g patent application 540,947 filed June 30, 1987 by Prober, et. al.
Four 1uocescent dyes were s~lec~ea with emi6~ion maxima at 505, 512. 519, and 526 nm. These maxima may ~end to 6hift 60mewhat when in the environment of gel elctrophoresis. ~he6e emis6ion characteristic~ were created by the appropriate chemical group subfititution6, 6uch a6 methyl groups, at specified loci in the parent compound (9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene).
Each of the four dyes prepared have reactive carboxy group~ provided by a sarco~inyl moiety covalently ~2~9~

bound to t~e 9-position of the parent compound, which are ~apable of Porming covalent attachment~ with amine groupfi i~ linking moieties that 30in th~ dye~
with selected nucleotide6. U6eul linkin~ ~oiQties found are a group o~ alkynylamine derivat~Yes which ~ontain a terminal amino group that can form covalent attachment~ with the dye carboxy g~oups. A preferred linker i6 a 3-aminopropynyl derivative whi~h is covalen~ly attached eo the 5-po6ition of ura~il (T) or cytosine (C). or to ~he 7-pvsition of deazaguanine ~d-G) or deazaadenine ~d-A).
Appropriate linker-nucleotide derivative6 or u~e in the ~ystem of ~his invent~on were prepared ~ieh 2~.3'-deoxyribo~u~leotide6. whi~h ~e ~own to ~er~e as DNA chain ~ermi~ati~g 6ub6erate~ for DNA
poly~era6e6. Ie ~as ~ound that covalent ~et~hment of the a~i~opeopynyl-~',3'-dideoxynucleotide8 to the ~luores~e~t dye~ in appropriate combination~, did noe sub6tantially d~mini6h the c~ai~ terminating propertie~ of the u~sub6tituted - 2',3'-dideoxynucleotides. ~he four dye-li~ke -dideoxynucleo~ide6 A,G,C,T ~elected are lllustrated by ~he ~truc~ures:

11 29~ 2 C~ CH~
o~O

N ~ ~ N H

o~ Pa ~y 2~) ~Xc~

2 5 c;~ ~j O~-~P~O~

7'7~

~Ha C~
~D~O

0 H~ NH~

t: H 3 ¦ ~ ~
09-~P~o~ 0 '`~X, ~' =~> H~
N ~ ~ O
C H ~

D3'~P~O N~[N~NH2 G ~

~9~

They were ~ound to ~erve as u6e~ul ~hain ~ermina~ing ~ub6trates for rever~e ~ran6cripta6e ~avian myeloblasto~i~ viru~) in a moditication of the well-known SangQr DNA sequencing method. The S alassical Sanger me~hod use~ a primer, DNA template, DNA polymerase I tXlsnow fragment), ~hree unlabelled deoxynucleotides and one radiolabeled deoxynucleotide in eac~ of ~our reaction ve~els that each con~ain one o~ four 2',3'-dideoxynucleotide~. which 10 ~orrefipond to the four I)NA base6 tA.C,T,G).
Appropriate reaction ~ondition~ are creaeed which allow the polymera~e to copy the te~plate by lg adding nucleotide6 to the 3~ end o the prim~r. A
~ultitude o~ reaction~ occur 6i~ultaneou61y on ~any primer copie6 to produce ~NA fragmen~ of ~arying length which all contain the radiolabel a~
appropriate nucleotide6 in each fragment, ~nd which al~o irreversibly terminate in one of the our dideoynucleotide6. Thi~ ~et of fragment6 i~
typically 6eparated on a polyacrylamide ælab electcophore~i~ qel in four lane~, one lane corresponding to each of ~he ~our dideoxynucleotide reaction ~i~eures. After the frag~e~ts haYe been ~eparated, a photographic fil~ i~ placed on the gel, exposed under aperop~iate conditio~s, ~nd a DNA
~equence i~ inferred ~rom reading the patter~ of banas created by the radiolabel on ehe ilm in order of thei~ appearance i~ the four la~eg from the botto~
o~ the ~el.
~ he ~odi~icat~on6 permitted by u6ing these ~ye-labelled terminator~ ~nolude omie~ing tbe radiolabeled nucleotide and substieu~inq the dye-labelled chain ~er~inator~ gor the unlabeled 2~,3'-dideoxy~ucleotides. Reaction ~ixture~
~actually a ~ingle reaction ~ember can be used) will now contain rag~ents which are labeled on their 3l end~ with a fluoropho~e ~hat correspond6 to each of four DNA bases. ~he reaction ~ixture(s) are combined and electrophorat~cally 6eparated. Sequence i6 infer~ed by the oeder of appearance o~ ~and~ being ce601ved in time or space that are revealed by the presence of fluore~cent radiation. Therefore, the ~luorescent dye-labelled dideoxynucleotide6 previou61y de6cribed are the preferred 60uece6 of clo6ely 6paced emitted radia~ion to be detected in ~he 6yste~ and ~ethod of thi6 invention. An alternative source of emitted radiation which can . e ` :c~ . j also be useful in the system and method of thi~
invention are the fluorophores described in the Smith et al. application. Their use would require selection of the appropriate laser frequencies and wavelengths of the preselected filters.
The optical arrangement for irradiating the lanes of tha electrophoresis slab 10 is shown in Figure 2. The system and apparatus of Figure 2 m~y be used with any fluorescent or other type system to distinguish between and measure the intensity of closely spaced emission radiation bands. However, it will be described by way of example of detecting the emissions from DNA fragments labeled with the particular reporters (dyes) set forth in the Prober et al. application. The dyes described in Prober et al. have peak emission wavelengths of abut 505, 512, 519 and 526 nm. It includes a laser 30 which is selected to provide an exciting beam of radiation 32, with a specific wavelength determined as a function of the excitation wavelengths of the fluorophores used. The specific source used with the dye fluorophores disclosed in Prober et al. is an argon ion laser with a wavelength of 488 nm and a 0.8 mm diameter light beam 32 operated at about 50 mW. The light beam 32 passes through an excitation filter 34 and is then directed into scanning optics 36. The filter 34 is selected to block out any undesired excitation wavelengths that could otherwise interfere with the detection process. However, for sufficiently spectrally pure lasers this filter may be omitted.
The scanning optics 36 include a prism or mirror 3~ mounted on a fixed support (not shown), an astigmatic focusing lens 40, a second prism or ` ! ' `
~, .

~29~

~irror 43, and a cylindrical optic ~upport 44 all ~ou~ted to the 6haft of a stepping motor ~6. The beam 32. upon entaring the ~canning o~tic~ 36 i8 ~ir~t directed downward by the pri6m 38 into t~e S cylindrical opening of the optical ~uppor~ ~4 and through the eOcu~ing len~ 40. Prism 3~ ~erves to direct ~he beam rom the lafier into ~he ~canning optic~ 36 thus facilitating convenient placement of the la~er 30. ~he lighe beam, pas6ing through the focusinq len~ 40 i~ concentrated into an ellietical spot, in a prefer~ed case o~ about 0.2 ~m ~ 1-2 ~ i~
~rog6-sectio~. The focu~ed llght beam 32 16 directed t~rough an exit apereure ~2 by the seeond pr~sm 13.
The opt~c support 44 i6 ~oun~ed ~o the ~hafe of the stepping ~otor 46 ~uch that by aetuating ~he ~teppi~g ~otor 46, the ~en6 40 and ~he prism 43 are rota~ed to cau~e ehe light ~ea~ 32 to an~ularly ~ca~ a horizontal plane perpendi~ular to the ~haft axi~ and to the plane o~ the ~el 10. -Thi6 l~ht beam 32 ~ 8 zo directed a~ the ~e~ection zone lg of the electrophore~is slab 10.
The light beam 32. u~o~ entering the slab 10 e~cites the reporter ~aterial, here fluorescent dye labelled DNA ~ragment6, a~ ~hey ~igrate through the de~ection zone 19, causing them to ~luore6ce at wavelenqth~ ~hi~ted from ~he axcitation wavel~ngth.
The peak e~i~sion wavelength~ ~or the dyes de6cribed i~ Prober et al. are about 505, 512. 519, and 526 nm:
~oweve~ the sy~tem i 8 adap~able to di6criminate wavelengths a~60ciated with other 6et6 of dye6 with closely 6paced emis6ion band6. YurthermoreO while a laser source i8 pre~erred ~ince it allow6 a ~inimum of extraneou6 filtering and optic6, other 60urce~
including a non-coherent 60urce 6uch a~ a xenon ~rc la~p could be u~ed.

~29~ 2 To increase ehe toSal radiant energy emitted ~y the f1uorescent specie6, a reflective surface 50 (Fig. 3) can be po6itioned oppo~ite from ehe excitati~n ~ource. In the pre~erred apparatu6, a 5 mir~or~d ~urfa~e i6 deposi~ed directly onto the outside of the o~ter plate 12 which ~upport6 or contain~ the gel. The excita~ion light 32 which i6 not ab~orbed by the emitting 6pecies con~inues through the plate 12 and i8 reflected back toward~
~he species by surface 50 to provide e~entially ewi~e the a~ount o e~cita~ion light. Addit~onally, the light ~ive~ ofL by the fluole~cent fragment~ i~
emit~ed ~n all direc~ion6 so that light dire~ed toward6 the r~flecti~e surface 50 16 reflected al60.
The net incr~a~e in 1uore6cent 6i~nal available for detectio~ ~ approxi~ately ~ ti~es the ~mount available without the re~l~ctive 6urface. ~he preferred ~eChod of providing a reflective surface i~
accompli6hed by coating Phe outside of the plate which ~upports the yel 10. Alternaeively, a mirror could be ~xter~al to the gla88 but the increa~ed ~umber of lnterface6 that the li~ht pas~es through would cause additional unde~irable scattered exci~ation light. The radia~t energy or light em~tted by the fluore~ent specie~ i6 collec~ed by two ~uitably positioned uppe~ and lower photodet~ctor ~odules 52 and 54, re~pectively. These detector module6 can be seQn ~ost clearly in ~ig. 3 in which the detail6 o~ their cons~ruction is 6hown.
In accor~ance with thi6 inven~ion, the ~odule6 52 and 54 a~e positioned above and below the plane of scanning of ~he light beam. The module6 are light tight in ~uch a way as tc eli~inate stray light not directly coming from the excitation region. Each ~odule ~ompri6e~ a photomultiplier tube tPMT) 56 of conventional type having a wide entrance area. A
suitable photomultiplier tube is the Hamamatsu R1612. Each module 52, 54 also has a separate wavelength selective filter 58 positioned between its ~MT 55 and the fluorescent species in the qel 61ab 10. The f ilters 58 preferably are cu~tom interference filters which may be ohtained from Barl Associates in Westford, ~A, which have complementary transmission band characteristics as ~hown in F;g. 4 and are pos;tioned to be transverse ~preferably perpendicular on average) to the light 60 emitted from the species. Thi6 positioning permits t~em to operate at optimum efficiency a~ will be described.
One ~ilter 58 having a transmi~sion charac~erist~c denoted by the waveform A of Fig. 4 is ~een to largely pas6 the lower emission wavelengt~s and reject the higher emis6ion wavelengths. The o~her filter 58 having a ~ransmission characteri~
denoted by the waveform B (Fig. 4~ does precisely the reverse--it largely pas~es ~he higher wavelengths and largely rejects the lower wavelengths.
Finally, each module 52, 54 has a transmission filter 62 positioned between the wavelength selective filte~ 5B for that module and the emitting species. These filters can be reversed. Each transmi~sion filter 62 rejects incident light entering the filter at off axis angles greater than a predetermined angle. The two wavelength selective filters 58 enable the system to distinguish between closely ~paced emission spectra.
Liqht impinging on these wavelength filters 5B will either be transmitted, absorbed, or reflected. The emission spectra of the four illustrative dyes selected, namely G505, ~512, C519, and T526, are illustrated in Fig. 4. The transmission filters 58 ~2~72 2q have been cho~en to have complementary tran~mi66ive characteri6tic~ corre~ponding ~o curYes A and B in Fig. 4 a~ de6cribed.
The two f ilters 58 are 6een to have loughly S complementary tran~iB~ion V6. ~a~eleng~h charac~eri~tic~ in ~he emis610n wa~elength regio~ of ~he four dyes, with the tran6i~ion ~avelengths occurrin~ near the center o~ ~iddle of the 6pecie6 radiant energy ~pectra. A~ the fluore6cence ~pectrum shift~ from the ~horte~ to longer wavelength~, the Latio of light tran6~itted through the upper f il~er 5a tin the drawi~g) to light ~ran~mi~ted ehrough lower filter ~8 I~ the drawing) cha~ge6 in ~
~ontinuous ~annerO The ~o~t 8ui~able il~er~ for 15 thi~ application a~e inter~ere~ce til~et~ ~hich have both a high relative tcan6~i~6ion and high ~loc~ing at the excitation wavelen~th. Although the~e pa~ticular filter~ have been cho6en to accommodate tbe parti~ular dye6 selected ~or thi~ applic~tion, a different ~et of dye~ could be ~uitably differen~iated with other ~ilter ~e~ ba6~d on these princlples.
~ he de~ectore and correspondi~g filt~r6 are selec~ed to have a rel2tively large area for this appl~ation. ~h~ detector6 56. preferably photomultiplier tu~es, have large entrance window6 nominally about 8 by 3.5 ~m. Ia this way~ a relatively large area lg on ~he gel ~lab, can be ~onitored wiehout the need for i~aging optic~ which inherently create ineffecieslcies in the light collection and ~re 60urce~ of scattered light. The detector6 56 in this 6ystem are po6itioned approximately 2-3 cm. from ~he emittinq ~pecie6 such that mul~iple sample~ can be continuou61y ~onitored 3s with high ligh~ collection e~ficiency. Under ~hese 7~:

conditions, it may be said that the detector6 have a large solid entrance angle.
~ s mentioned above this ordinarily would lead to the transm;sslorl oE undesired high angle off normal (to the de~ectors) scattered excitation light. This occurs because at off-normal angles, the path length t~rough the deposited layers (cavities) of the filters 5~ is changed siqnificantly, thereby 6hif~ing the filter characteri6tics toward the sho er wavelengths. (Changes in bandwidth and the peak transmission are minimal for small incident angles with respect to the normal to the filte~s.) Since the sample emits radiation from the detection zone in ~11 directions the detection of 60me off-axis radiation is inevitable. Light impinging on the ~ransmission filters 58 near the normal angle i~
desirable, but using large detectors spaced a fixed distance from the emitting species allows ~cattered excitation light as well as fluorescent emitted light to impinge over the entire surface of the filter at relatively oblique angles. This light is then transmitted through the filter independent of the specified filter characteristics.
In accordance with this invention, this problem is ~olved by the use of ~pecial transmission ilters 62 coupled with the wavelength filters 58 such that the light impinging on the wavelength filters 58 is limited to a fixed range of angles close to the normal direction. The majority of the light which i~ impinging at an angle greater than a cut off value is either rejected or absorbed. If additional of~-angled rejection is necessary, this invention can be combined with appcopriate baffling, the use o~ colored filter glass or other known means.
3s ~L2~7~

A known device 62, u6e~ul ~or the tran~mi~6ion filters ~2, having characteri~tic~
capable o~ eejecting o~-angle light, i~ ~anuactured by XNCOPq lo~ated in Southbridge, Ma~. This device 5 consi~t~ of a tightly paeked bundle of optic f iber~
fused together, each ibee ha~ing a dia~eter of apps~ximately 10 ~ic~ons. The bundle i6 CUlt e~a~6ver~ely acros~ the ~ibers to produoe an opei~
ele~ent o a desired thickne6~. Since ea~h one of lo the ~iber~ in the element ~a~ a nominal numerical aperture o~ a~out 0.35 only light i~pinging within a 2~ angle i~ allowed to be tran6mitted. Light impinging at a~gles greater than the ~ccepta~e a~gle is either re jected by f irst ~urace ref lectio~ or i6 ~s pa~sed through the claddi~ of the tran~mi6~ion f iber. Light which pas~e6 ehrough the ~ladding ca~
co~tinue through the next ad jaeerlt f ber in the ~ame ~anner to ~ ally rea~h the opposi~e 6urface of the plate and exit at a~ angle similar to the original 20 angle of incidence. ~hi6 parti~ular light wi 11 impinqe on the wavelength f ilter6 58 in each ~odul~
52, 54 at an und~6irable an~le and avoid the de6igned ilter characteristics. To eliD~inate ~he occurrence of this eype o~ light erans~i6~ion through e~e f iber 25 plate, 3-6t ~e3ctra ~ueal ab~orberN (EHA) fibers are e~venly disper~ed throughout the bundle. The nocmal di~tLibution of these f iber6 create~ all absorbing bacriee tha~ dramatically attenuates light which pa~se6 through the ~alls of the optic ~ibers but doe6 not affect the internally ~eflected lig~t tAa~
propagates ~hrough each ~iber in the plate.
Returning to Figures 2 and 3, ligh~
tran6mitted through the re6pective filter ~yBtem~
62-5~ i6 directed to the respective detectors s6.
The electrical signal6 from the detector~ are ehen . .. . . . . . . .. . . .... . .. . . . . . .. . ... . .

~299~2 passed via rcspective preamplifiers 66,68 and to respective analog to digital ~A/D) converters 70,72 and thence to a system controller 80. The task of the system controller Bo may be performed by a small computer such as an IBM PC. A function of the system controller 30, which is de6cribed by the flow diagram Figs. 4~, 4B and 4C is to compute the ratio of the two signal functions (the emission intensities on the PMT~s 5~ for each d;{ferent emi6sion wavelength) among other conCrol tasks. The wavelength filters 62 modulate the intensity of the signals in each of the differen~ wavelength regions according ~o wavelength, i.e., fro~ one detector, the shorter wavelength emissions will have a lower ampli~ude ~ignal ~alue and the longer wavelength emissions will have a higher amplitude. The reverse is true for the other detector since the filter~ have complementary characteristics as described.
Thus, as a particular species, i.e., a dye-labeled DN~ ~ragment, following separation in space in the yel slab, passes through the detection zone 19 o~ gel slab 1~, its emission spectra will generate two signals, one at the output of each detector 56, that vary in amplitude as a function of the emitted wavelength and time (because of the movement through gel slab 10). The amplitude modulated light signals are at thi6 point converted to electrical signals which are then digitized for such processing as desccibed. After conversion, the functions of the two digital ~ignals are ratioed, i.e., to obtain the quotient of the first and second signals of {luorescent light for each fluorophore.
The maynitude of the ratio signal is indicative of the identity o~ the species. Actually the ratio signals amplitudes for each dye tends to fall into distinct clusters or g~oupings which aee readily distinguishable.

use~ herein the term "clo~ely 6pacadl' in ~ecm6 of the emi~sion chacacteristic~ of the dyes or fluorophore~ i~ a ~omewhat relative ter~. The ~inimum spacing between the center o~ e~ls6~0n o~ the S dye6 i6 large enough ~uch ehat the diffeeence in ratio6 of signals ~rom the two deee~tor~ ~or any two adjacant dyes i6 distinguishable above the inheren~
~ystem noi~e.
In order ~o improve dye de~ection selectivity, the filter characteri6tic6 can be urther optimized. Thi~ can be accompli6hed by choo6ing filter6 with ~haraeteri~ti~6 that change 6ubstantially over the diferent dye emis6ion ~pect~a. However. eo distinguish a closely 6paced group of dye~, it i~ preferable to ~ave ~elatively sharp ~ilter tran~itionl; that occur ~ear the center vavelength of the group of dye emi6~ion~ in order to evenly distribute ~he ~hange in ra~io of 6ignals in the two filters ~or the diff~re~t emi6~io~ ~pectra.
l~he ~haracteri~tics of an individual ~ilter can ~1160 be ~i~e-~uned to a degree by ~lightly varying the angle o~ lnciden~e of the fiber optic facepla~e output flux relat~ve to the inter~Eerence ilter normal due to the i dio~yn~racies oî i~t~r~erence ~ltees a6 di~u66ed æreviou61y.
In ~any ~ase6, the extra ~ural adsor~tion of the t~an6mi6~ion ~ilter 62 need be u6ed ~or the wavelength filter having a pa~band clo6est to the exsitation wavelength o~ the la~er needs to be ~ore than ~he other tranfi~i66~0n filter. In this exa~ple where the exciting lafier operate6 at 4~8 ~m, the wavelength filter 5~ having the pa6sband A (Fig. 4) requires a trans~i6sion ~ilter 62 with twice ~he EMA
as used with the wavelength filter 58 having the pas6band B (Fig. 4). In ~ome ca6e6 only one tran8mission ~iltere i~ needed.

: - .

The 8y6tem controller 80 convert6 the ~igital signal6 received from the A~D converter6 70 and 72 into DNA ~equen~e informatio~. In ~06t Ca6e~, ~hi6 will be done by a co~pu~er executing pro~ra~ in eeal eime. ~hi~ means ehat ~ata i~ proce66ed and sequence information i6 determined ~oncurrently wi~h ehe aequisition of raw da~a f~om ehe detec~ors.
Con~eptually, the operation of ehe 6y6~em lo controller may be broken down ~nto three lnteractiag pro~e6~es: data acquasition or ~nput, da~a analy6is, and output. The proces6es interact ~y 6haring daea and by s~aring eiming infor~ation which keeps the~
~in 6eep" ~nd prevent~ ~hem fro~ ineerfering with 3ne another. ~he detail~ o~ how the6e interactions ~re a~compli~hed depend on the language and hardware ~ho6en ~nd i~ no~ o~ funda~ental con~ern.
The data ~qui~ition and processing 60 per~o~ed to obta~n DNA base ~equence ~or~atlon ean be best under~tood by re~errîng to e~e flow char~6 i~
- Figures 5~ 5BD and SC. ~he~e figureE repr~en~ a general ~ethod by whieh the r~w detector input ro~
the ~canning 1uorescent detection 6ystem ~ay be ~onverted into output, i.e.i ~he DNA ~quence o~ the Z5 ~ample. In thi~ di~cu~sion the followi~g ter~s ~hall ~ean:
1. i iB the index o~ the ~urrent data point being acquiced. This point i~ acquired at ~ime ~(i) min.
2. k is the index o~ the current data point being proces6ed taken at t(k)min. In a yeneral data processing scheme, k need not equal i, i.e. data processing ~ay lag behind data acquisition.

2g , ... . . . .. ... . . .... . .... . . . . . . . . . . .

` ~l%9~72 3. tti) is an array o~ ~ime points at which data was acquired. Example: t(5),6.2 min would indica~e the 5th data point wa~
acguired at 6.Z minute& afte~ the start of the run.

4. Dl(i) is the array of data from the ~i~$~ detec~or.

5. D2(i) is the array of data from the second detector.

6. J is a ~ount of peaks detected.

7. N is the number of data points across a given peak.

8. m i8 an index of points acro6s a defined peak in either Dl(i~ or D7(i). m=l at the ~tart of a peak; m=n at ~he end of a peak.

9. W is a function (e.g. the ratio) of the detector output6 Dlti) and D2(i), the value of which determines the identity of the DNA
base corresponding to a given peak.
D~TA ACQUISITION
A general data acquisition process for a single channel is shown by the flow chart 5A. The index i, which points to the current acquired data, is initialized. The program accepts an input which determines how long the run will take, i.e. the total number of data points I[tota~ fter the ~aw data arrays Dl and D2 are initialized, the process enters an acquisition loop. Data are read from the detectors, digitized, and placed in the arrays as Dl(i) and D2(i) for detector 52 and detector 54, respectively, acquired at time t(i). (For the purposes of this discus6ion, the two readin~s are simultaneous.) At this point, the index i is inc~emented and compared to I(total~. If i is less - ~2~72 ~ 1 than I~tal), the acqui~ition loop i6 repeated.
If i e~ual~ I(total)~ the run i~ 6topped. In a more elaborate schame, ~he program ~ould ~ense when to end the run automatically by mea6uring 6everal kerf~rmance parameters (such as signal/noise ratio, peak re~olution, or uneer~ainty in a~igning ba6es) at eaeh peak of the ~un. If a combina~ion o such actor6 ~ailed to ~e~e preset criteria. the run would be terminated by the ~omputer.
The primary data input i~ the raw data from the detectoss ~nd ~he output is 6tored in the data ~rray6 Dl~i) and D2ti) ~hich are ~haeed beeween ~he acqui6i~ion and the da~a analy6i~ procesEe Thi~
~che~e i~ dep~ted s~hema~i~ally in Figure 5B.
Alehough the two program~ run independently and si~ultaneou~ly. ~o~e control information ~ust be ~assed between them in order ~o maintain proper tim~ng. ~or example, the proce~sing 6tep~ cannot be allowed to overeake tbe acqui6ition 8tep becau~e ie would the~ be ~ttemptinq eo proce~s nonexi~tent data.
ANALYSIS
~ he data proce~sing algorithm depicted i~
~iqures 5~ and 5C i~ an exa~plQ of a qenecal 6che~e to dete~t and ~den~ify dye-labeled ~pecie6. It i~
not ~eant to be all-inclu6ive. ~ather, it illu~trates the primary feature6 that are neee66ary in developinq any real analyzer program and i6 exemplary of apeli~ant6' preferred embodiment.
~fter lnitializing the proees6ing index k (as distinct from the aequi6ition index i), the proqram enter~ a 6imple loop which read6 data Dl(k) and D2(k) from the data arrays provided by the acqui6ition proce66. The program then ask6 whether ~he curren~ poin~ i8 on a peak. A number o~
algori~hms exi~ which ~an determine thi6 ~ondition;

t72 details are not needed here. I~he term "peak" is meant in a general sense. A peak in Dl will generally be accompanied by a peak in D2. However, depending o~ the identity of the dye, the peak6 in these two channels may differ considerably in intensity. They will, however, coincide in time.
Thereore, a weighted average of the tWG signals, the ~tronger of the two ~ignals, or some other combination of Vl~k) and D2(k) could be used to define a "peak'~ in time.
If the ~urrent processed point is not on a peak, the index k i6 incremented ~nd compared with the acquisi~ion index i. lf k equals I(total)o the run is over and the program ctops. If ~ i8 less than i, the next data point~ are ~etched from the arrays Dl and D2 and the loop executes again. If k is equal to i, it means that processing has caught up with ~he data acquisition. In ~his event, the proce~sing program waits a small period of time (typically a second) and again tests the values of k and i until processing can resume.
If the current processed point is on a peak, the index m is incremented. Index m counts the number of points across the current peak. The values Dl(k) and D2(k) are placed in temporary array called Dlpeak(m) and D2peak(m), respectively. The program then tests whether the current point i~ the last point of the peak. lf ~his is not the last point on the peak, program control returns to the upper loop which increments k, tests its value against i, and reads the next pair of data from the arrays Dl and D2.
I the current point is the last point on the peak, the peak counter is incremented and the program proceeds to determine the identi~y of the peak. The re~ult is the identity of ehe next base in ~9~

the ~NA 6eguence. The program calculates the ~unction W foc the current peak as described above, using the arrays Dlpeak(m) and D2peak(m) as input data. Eac~ nucleotide base will have as60ciated with it a pair of peaks which give a characteristi~ W.
Thus, based on the value of W for ~hic peak, the pro~ram givea as ~utput the DNA base identity A. T, C, G. The peak point index m and the arrays Dlpeak and D2peak are rese~ to ~, and the program again entQrs ~he upper data acquisition loop as ~hown in Figure 6B.
The above 6cheme may be extended to a multi-sample ~can~er. In a multiple-channel instrument, the laser beam would be moYed to the nex~
sample position before re-entering the data acquisition loop in Fig. 6A. In addition, separate data arrays (for example DlA(i), DlB(i)...DlKi) and D2A(i), DZB(i)...D2K(i) for lanes A, B,...K) would be assigned to each lane or sample position. Data acquisition and processing would proceed for each lane in turn in a manner similar to that described above.

Claims

1. In a system for detecting the presence of radiant energy emitted from different species, following separation and the identity of such species, having first detection means responsive to the radiant energy emitted by of the species for generating a first signal that varies in amplitude in a first sense as a function of the nature of the species, second detection means responsive to the radiant energy for generating a second signal that varies in amplitude in a second sense different than the first sense as a function of the nature of the species, and third means responsive to the first and second signals for obtaining a third signal corresponding to the ratio of functions of the first and second signals, the amplitude of the third signal being indicative of the identity of each of the species, the improvement wherein the first and second detection means each include:
a detector having a large solid entrance angle positioned adjacent the species to receive radiant energy emitted from the species for generating one of the first and second signals, a wavelength selective filter means positioned between each respective detector and the species, each wavelength filter means having transmission vs.
wavelength characteristics that are complementary, and wherein one of the first and second detection means includes a transmission filter means for rejecting radiant energy incident on the transmission means at an angle greater than a predetermined value.

2. A system as set forth in claim 1 which also includes a laser adapted to direct an exciting beam of radiant energy, through a propagation region, to the species, said species having an excitation region, the laser beam of radiant energy having a wavelength lying within the excitation region of the species.

3. A system as set forth in claim 2 wherein the transmission filter is associated with the wavelength filter means having a passband closest to the wavelength of the laser beam radiant energy.

4. A system as set forth in claim 3 which includes means adapted to separate fragments of DNA
or other molecules labeled with emitting species of materials, positioned to be excited by radiant energy from the laser.

5. A system as set forth in claim 2 which includes means adapted to separate fragments of DNA
or other molecules labeled with emitting species of materials, positioned to be excited by radiant energy.

6. A system as set forth in claim 4 wherein the first and second detection means are positioned on opposite sides of the region propagating the laser beam of radiant energy.

7. A system as set forth in claim 1 which includes means adapted to separate fragments of DNA
or other molecules labeled with emitting species of materials, positioned to be excited by radiant energy from a laser.

8. A system as set forth in claim 2 wherein first and second separation means are positioned on opposite sides of the region propagating the laser beam of radiant energy.

9. A system as set forth in claim 8 which includes means to sweep the laser beam of radiant energy across the separation means to excite the species in sequence.

10. A system as set forth in claim 9 wherein the wavelength selective filters have a transition in their transmission vs. wavelength characteristics centered at about the middle of the species' excitation region.

11. A system as set forth in claim 10 wherein the transmission filter has an extra mural absorber among plural optical fibers positioned to have parallel generatrices transverse to the first and second detectors.

12. A system as set forth in claim 11 wherein the predetermined value of the rejecting angle of the transmission filter is about 22°.

13. A system as set forth in claim 1 wherein the first and second detection means are positioned on opposite sides of a region containing a beam exciting radiation.

14. A system as set forth in claim 13 which includes means to sweep the beam of exciting radiation across a separation means to excite the species in sequence.

15. A system as set forth in claim 14 wherein the wavelength selective filters have a transition in their transmission vs. wavelength characteristics centered at about the middle of the species' excitation region.

16. A system as set forth in claim 1 wherein each detection means has a transmission filter means.

17. A system as set forth in claim 16 wherein the wavelength selective filters have a transition in their transmission vs. wavelength characteristic centered at about the middle of the species' radiant energy spectra.

18. A system as set forth in claim 18 wherein the transmission filter has an extra mural absorber among plural optical fibers positioned to have parallel generatrices transverse to the first and second detection means.

19. A system as set forth in claim 1 wherein the transmission filter has an extra mural absorber among plural optical fibers positioned to have parallel generatrices transverse to the first and second detection means.

20. Apparatus for detecting the presence of fluorescent radiation derived from fragments of DNA
or other molecules labelled according to type with different fluorescing species of materials comprising:
separation means adapted to specially separate the molecules, means including a laser to sweep in a frist plane a beam of radiant energy across the separation means to excite the species, first and second photodetectors having large solid entrance angle positioned on opposite sides of the first plane to convert fluorescent radiation emitted from the species into first and second signals, first and second wavelength filters having complementary transmission - wavelength characteristics interposed between the respective photodetectors and the sepraration means, a transmission filter, interposed between one of the respective photodetectors and its separation means, for rejecting radiation incidence on the photodetector at an angle greater than a predetermined value, and means responsive to the first and second signal for deriving a third signal corresponding to the ratio of functions of the first and second signals, the third signal being indicative of the identity of the fluorescing species.

21. Apparatus as set forth in claim 21 wherein the wavelength selective filters have transitions in their transmission vs. wavelength characteristics centered at about the middle of the species fluorescent radiation.

22. Apparatus as set forth in claim 22 wherein the transmission filter has an extra mural absorber among plural optical fibers positioned to have parallel generatrices transverse to the first and second detector means.

23. Apparatus as set forth in claim 24 wherein the predetemined value of the rejection angle of the transmission filter is about 22°.

24. Apparatus as set forth in claim 21 wherein the transmission filter has an extra mural absorber among plural optical fibers positioned to have parallel generatrices transverse to the first and second detector means.