CA2251809A1

CA2251809A1 - Method, apparatus and kits for sequencing of nucleic acids using multiple dyes

Info

Publication number: CA2251809A1
Application number: CA002251809A
Authority: CA
Inventors: Thomas J. Digby; Alexandre Izmailov
Original assignee: Individual
Current assignee: Bayer Healthcare LLC
Priority date: 1996-04-18
Filing date: 1997-04-16
Publication date: 1997-10-30
Also published as: EP0895544A1; US20030003498A1; US6440664B1; WO1997040184A1; AU2500197A; US6432634B1

Abstract

An instrument for sequencing oligonucleotides is loaded with the products of four sequencing reaction mixtures. These products are a combination of A, C, G and T reaction products for several sequencing reactions. The products of the different sequencing reactions are labeled with fluorescent tags which are distinguishable one from the other on the basis of their excitation or emission spectra. After separation of the oligonucleotides by electrophoresis, the order of the detected peaks is used to call the base sequence.

Description

L~ L~ J_ ' i ~ ' CA 02251809 1998-10-13 MET~HOD, A~PARi~TUS f'.N~ KITS ~OF~ SEQ~JENCING OF
~JCLEI~ AC~:)S USING MULTIPLE l:)YES
.
DESCR~TIOI'~

BACKGROUND OF THE IN~ENl'ION
Thi~ application relates to an improved me~hod for se~uencing of nuc~eic acid~ using mt~ltiple fluoresce~lt l~bels, and to ~rpa-~tu~ and kits adapted for use witl~ the metho~
Sequencing of nucleic acids usin~ the ~13in terrnination method in~olves the general steps of combinin~ th~ target nncleic acid polymer to be sequenced with a sequenci g pnmer S which hybridizes with the tar~et nucleic acid polymer; e~tendir,~ the sequer.ci~g ~ r in ~he presencc of nor~nal nucleotide (A, C, G, ~d 'r) and a chain-termina~g nucleotide, such a~ a dideoxynucleotide, which pr~ents further extension of the pnm~ o~çe ircorporated; and analyzing the pr~duct for the le~gth of the ~xtended ~ra~ncnts obtained. Analysis of fra8ments may be done by electrophoresis, for example on a polyacrylamide ~el.
Alth~ugh this type of analy~is was ori~n~Lly ~ Çv.IL~çd us~ng r~diolabeled fr~ t~
which were dc~ectcd by autoradio~raphy af~er separation. modcrn automated D~ sequencers generally are desi~ned for llse wi~h sequencing fr~gmente having a fluore~c~ll label. The fluorescently labeled fragments are detected in re~l time ~lS they m~grate past a de~ector.
US Patent No. 5,17 L ,534 ~hich is incorpora~ed herein by reference descnbes a Yariation of Ihis basic ~e~u~ncin~ in which f'our dil~ferent fluoLcsce~t labels arc enlployodl one for each sequencing reaction. The fr~ nt~ dcvelop~d iII the A, G, C and T
se~uenci~g re~ctions are lhen recombincd a~d intr~duced together ~nto a s~p~tion matrix. A
sy~tem of optical filters is used to indi~idually detect the fluorophores as they p~ss the detectt-r. This allows the t~roughput of a se~u~ncing apparatus to be incre~sed by a factor of 20 four, sincc the four sequencin~ reacti~n which were previously mn in four separate laneg or capillaries ca~ now be nln in c~e.
Olesen ct al., "Chemiluminescent DNA Sequcncing with M~l~sp~ex Labe~ing", Bio~eckniques I5 480-485 (1~9~) describe a mo~lifir~tior of a S~n~er sequencing techmquc in which several samples (e.g. ~our samples) arc sepa.~tcly used as templates for the 2S production of sequencing fragrncnts. Each sample was reac~ed with a different primer species labeled w~th a ~irf~le~l~ cherr~~ n~scent hapten. Tlle A reactions from each sample were A~lENDED C5~c~T

L~ ) ' . L <~ CAl ' o i 2 ~ 18 0 9 19 9 8 r ~ )' )'J I ~ ; jr ~

then c~ bi~ed, 3nd lilcewise the C re~ctions, the G reactions a~d the T r~ o~s. The comb~ned r~ were t~e~ separated by e~ccucl horesis, ~r.sfe~e~ a~er sep~tinn tO a ~ylon membrane and sequen~ially observed using the differe~t cherni~llminescent labels. I~e signal strength for each detection d~creases with e~ch successive label ~nte~o~ated It is an objec~ of the present 1nvention to provide a fi~er improveme~t for use w~th chain terrnin~ion sequencing ,.,r~ti~n~ which c~ increase the ttlroughput of an ~s~lu,.l~L~l.

A~,~,E~ LLT

- -CA 02251809 1998-lo' i3 ~I L .~

SUM~IARY OF 7HE ~ TION
~n order to use nuclçic ~cid se~uencing as a dia~n~stic lool, it will ~e necess~ry to detumine the sequence ~f the s~me DNA re~ion from many samples. ~he present inven$lon rnake~ it possible to increase the throu~hput of an instrument being used for this purpose.
Thus~ a first ~specl of the invenlion prov,des a method for evaluating the s~quence of a ~r~es nucleic ~cid polymef in a plurality of samples. In this meth~d, each ~mple is f~rst di~,ided into four aliquots which are con~bined w~t~ four se~encin~ .eaction mL~ res. E2ch sequencing rcaction m~xnlre contaiIls a pol~n~erase en2~e, ~ Fnmcr for hybrid!~ing urith the t3rg~t ~ucleic acid, n~cleotide feedstoc~s and a different dide~xynucleotide. This results in the 10 form~t~nn o~an A-n~ Lu~, a G-mixture, a T-mixnlre and 3 C-mixture for each sample cc~aiRir.g ~ro~uct oligon~ ou~ i~a~ments of varying lengths. The product oligonucleotide fr~ n~s a,re labeIed vvith spectro~copically-delectable Lag, for example a fluore~ce~t ta~, and thesc tags will gencrally be the ~am~ for all four scquencing r~cti~7ns for ~ sample. However~
the spectroscopically-~etec~lble ~ags used for each s~mple 3re distin~uishable one ~om the 15 other on the ~asis of th~r absorption, excitalion or emi~sion ~pec~a.
Next, the .~-m~xtur~s for e~ch sample are combincd to form ~ combincd A mixrure, the G-mixtures are combln~ to fonn ~ combincd G-~ixhlre ~nd so on for all four miYrures. The c~mbilled mixturcs are loaded onto a separati~n ma~ix at s~aratc loading sites and an electric field is applied to cause lhe product oli~o~ oti~e fiagments to migr2le wit}lin Ihe separation 20 matnx. The sepa~ed product oLigonucleotide fragments haYi~ ifr~ t sp~,koscopica3 y~esectablc tags are ~ct~cted as they ~rate within thc separation mal;r~x.
- 'rhc mcthod of the invention c~ be used as describc~ above to determine Ihe positio~
of evcry ~ in the sec ~ c~, ar it call be uscd t~ deterrnine the pGSitiOIl of less than ~1~ four ~ascs. For example, thc method can be used to detcm~in~ thc pasition of only the A b~ses 2~ wi~i~ a s~q-lR~l~e for some diagnostic applications.
A fi~her aspec~ of the prese~t inventio~ is a kit usefill for diag~lostic $~1l~r~rin~ of a selected portion of a gcne. One ~mbo~imellt o~ such ~ kit conta~ns a plurality of s~lu~ncing primer~ f~r the sPl~ct~d portion of thc genc. each s~uencin~ prirner bcing identicaL in its ~IA
sequence but being labeled wi~ a cli~.~ spectroscopically-detectable ta~.
A f~rther aspcct of the invention is an appa~atus for perf~rrni~g the metho~ of the in~eneion~ Such ~n apparan~s compnses t~ CO S~

CA 022~1809 1998-10-13 WO 97/40184 PCT/CA97/002Sl (a) means for providing excitation energy to a detection site within a separation matrix disposed within the apparatus;
(b) means for detecting light emitted from fluorescently-labeled oligonucleotidefragments located within the detection site;
(c) configuration control means, operatively connected to the means for providing excitation energy and the means for detecting to provide combinations of excitation wavelength and detection wavelength specific for a plurality of di~l ~nl fluorescell~ly-labeled oligonucleotide fragments; and (d) data processing means, operatively connected to the configuration control means and the means for detecting for receiving a signal from the means for detecting and ~signing that signal to a data stream based upon the combination of excitation wavelength and detection wavelength set by the configuration control means.

BRTFF DF.~CR~PTION OF THE DR~WINGS
Figs. 1 A and B shows a schematic representation of the method of the invention;Figs. 2 A, B, C, and D show excitation and emission spectra for theoretical sets of useful fluorescent tags; and Fig. 3 shows an apparatus for evaluating the sequence of nucleic acid polymers using the method of the invention.
DETA~T F.n DF.SCRTPTION OF THE INVENTION
Fig. l A shows a schematic representation of one embodiment of the method of theinvention. The figure depicts the application of the method to two samples for clarity. As will be appal-enl from the discussion below, however, the method of the invention is not limited to two samples, and is in fact preferably applied for four or more samples, up to a limit imposed only by the number of di.ctingllish~ble tags which can be identified.
As shown in Fig. IA, two samples, "sample l " and "sample 2" are each divided into four aliquots and these aliquots are introduced into sequencing reactions Al, Cl, Gl, and T1, and A2, C2, G2 and T2. Each sequencing reaction contains the reagents necessary for producing product oligonucleotide fr~gmPnts of varying lengths indicative ofthe position of one-base within the target nucleic acid sequence. These reagents include a polymerase , . . .

I L . '~ L _ J ' CA 0 2 2 5 1 8 0 9 1 9 9 8 - 1 0 - 1 3 . ~
enzyme, for ex3tnple T7 polymesa~e, Seque-l~seTU, Thermo SequenaseT~, or the Klenow fr~ nt of DNA polyln~l~se, A, C, G ~ T nucleotide feedstocks; one typc of ~hain tcrminaîing dideoxynuclcotid~; and a s~quenc~ng primer.
A~er Ihe product aligonl~cleotide fragments are formed in each reaction mlxture, the prodlucts from reaction mixturc A I are combined with ~h~ products frorn reacti~n mixture A2 to form a c~mbined mixlure 10 which is loaded onto lane 1 of ~ sep3~tion matrix. Likewise, the products from reaction mixturc C1 are c~mbined with the prodlIcts from rea~;tion mixturc C2 to forrn a comhine~ mixLure I 1 which ~s loaded onto lan~ ~ ~f the separation matrix: the products from reactioll ~ixnlre Gl are combined with the prad~cts from re~ction mixture G2 10 to forrn ~ combined mixture I2 which is !oaded onto lanc 3 of the s~paration matI~x; and the products from reaction mi-.ctur~ T1 are combin~ ~ ~lie ~r~e;~ m reac..o.. i.~ re r to fonn a combi~ed mLltt~re 13 whic~l is ioaded onto lane 4 of tbe scpara~i~n rna~ix.
The key to the pre~ent in~entin~ thc use of l~bels i3~ the reactia~s A 1, ~ l, G l, ~nd Tl which are ~ tn~ ab~e from the labels used in reac~iuns A2, C~, G2 and T~, respec-tively. Thus, u~like the method ~escnbed in US Pa~er~ ~lo. S,171,534 where the labels us~d for lhe A, C, G. and T r~ction~ for a sample are distinct~ in the present inveneion ~he labels used f~r the follr sequenci~.~ re~ctions for any one samplc can be, and prefierabi~ are, the same. Tn~tearl, it is the labels whlch are used in the seueral samples which are distinct in the method ~f the insres~tion~
A~ ~ltem~tive ~mbodimeDt ofthe inventlon is illustrated in Fig. IB. I~ this case~ the ~p~ r wa~ts to sequence a plurality of genes (or di~ferent exorls of the same gene~ from one patient sample, The s~nple ~0 is divided into faur aliquots. A sequç.~ ng re3ction mix co.~t~ining the reagents n~ceCc~ for prodncing p~duct oli~m~c1eo~lde ~ nents of ~ng leng~hs is'ad~ed ~o each ~liquol. 1 he sequencing n~ix added to a first aliquot contains all the re~ for an A tesmination rcaction, plus ~ plorality of sequ~ g pri~ners, each onc labcled with a rliet;ng~ h~7~1e llu~yhore, ~nd eaeh one being specific for a ~ rt~ gene (or dif~.c.~t xon of thc same gene). ThC scquencing mix added to d se~ond aliquot con~in~ all of the reagents for a C termi~ation reacticn, plus thc same plu~lity o~'se~ nc;n~ pnmers.
Sequ~ r~act~o~ mixes for (i a~d ~ are made in the same fashion. These sequencingmixtur~ are rea~ted to produce oli~ntlllcleotides frag~slen~;, ~d the., loaded onto lanes 21, 22, 23, and ~4 of a sequ~ ng gel and s~pAr~t~d Us~ng t~s technique, any number of genes or CA 022~1809 1998-10-13 W O97/40184 PCT/CA97/002Sl exons in a sample can be ~imlllt~neously sequenced up to the limit imposed by the number tin~lish~ble tags which can be identified.
Suitable labels for use in the present invention are fluorescent tags. These can be incorporated into the product oligonucleotide fragments in any way, including the use of 5 fluorescently-tagged primers or fluorescently-tagged chain terminating reagents. Colored dyes detected using absorption spectroscopy can also be employed.
The fluorescent tags selected for use in the present invention must be dictin~ h~hle one from another based on their excitation and/or emission spectra. For example, as shown in Fig. 2A, a set oftags could be selected which had overlapping emission spectra (Eml, Em2, Em3 and Em4) but separate and ~listinglli.~h~ble excitation spectra (Exl, Ex2, Ex3, and Ex4).
A set of tags could also be selected which had overlapping excitation spectra but separate and distinguishable emission spectra as shown in Fig. 2B. Further, as shown in Fig 2C, a set of tags could be selected in which some of the tags have overlapping excitation spectra (Exl and Ex2) but separate and ~listingllish~hle emission spectra (Em] is ~ tinglli~h~ble from Em2), 15 while the others have separate and distinguish~ble excitation spectra (Ex1, Ex3, and Ex4) but overlapping emission spectra (Em 1, Em3 and Em4). A further combination of excitation and emission spectra is shown in Fig. 2D.
Examples of sets of suitable tags, together with the wavelength maximum for the excitation and emission spectra are shown in Table 1. Many other fluorophores are available 20 that can be used as labels for DNA sequencing reaction products. Such dyes are available from Applied Biosystems, Inc. (Foster City, CA), Molecular Probes~ Inc. (Oregon) and others.

CA 022~1809 1998-10-13 Table 1: Fluorescent Dye's suitable for use with the invention Fluorescent Dye Excitation Max (nm) Emission Max (nm) Texas Red X 599 617 Carboxy-X-Rhodamine 585 612 CarboxyFluorescein 494 521 CarboxyTetraMethylRhodamine 561 591 Carboxycyanine 5.0 650 667 Fig. 3 shows a basic layout for an appa- dllls for ev~ ting the sequence of nucleic acid polymers using the method of the invention. Light from a light source 31, which may be for example a laser, a light emitting diode, a laser diode, an incandescent or polychl o,..alic lamp, or any combination of such sources, is passed through an optical filter 32 if necessary to select 5 an approp.iate excitation wavelength which is directed to a detection site 33 in a separation matrix 34. Additional optical components, not shown, may be included as necessary to direct light to the separation matrix. Light emitted by fluorescent tags in the detection site 33 passes through a second optical filter 35 to a detector 36. Again, additional optical components can be included to direct light from the separation matrix 34 to the detector 36. Either or both of 10 the optical filters 32 and 36 may be adjustable under the control of a microprocessor, minicomputer or personal computer 37 to provide various configurations of excitation and emission wave]engths as di~cussed more fully below. The output from the detector is then transmitted to a data processing system such as a dedicated microprocessor, minicomputer or personal computer 37 for anaiysis to produce a report on the sequence ofthe sample being 15 evaluated.
In the case where the properties of the selected tags are of the type shown in Fig. 2A, the optical filter 32 may adjustable, for example by rotating several di~e. c~L filters through the path of the excitation beam, to produce excitation beams corresponding to the different CA 022~1809 1998-10-13 excitation wavelengths of the tags. An acousto-optic tunable filter of the type employed in U.S. Patent No. 5,556,790, which.is incorporated herein by reference, can also be used to - separate excitation wavelengths. Optical filter 35 may then be simply a cut-off filter selected to exclude light of the excitation wavelengths from the detector. Information concerning the position of the optical filter 32 as a function of time is tr~n~mitted to the data processing system, and used to permit intel yrelation of the fluorescence data. Thus, when the optical filter 32 is in a position that corresponds to the excitation spectrum of the tag used to label sample 1, the data processing system interprets the emission intensity as data for the sequence of sample 1, when the optical filter 32 is in a position that corresponds to the excitation spectrum of the tag used to label sample 2, the data processing system inle, ~ ts the emission intensity as data for the sequence of sample 2 and so on for as many di~el e-~L tags are used.
In the case where the properties of the selected tags are of the type shown in Fig. 2B, the optical filter 35 is adjustable, for example by rotating several dirrerenl flters through the path of the excitation beam, to selectively collect emissions wavelengths corresponding to the dirrel ~,l tags. Optical filter 32 may be simply a cut-off or band-pass filter selected to exclude light of the emission wavelengths from the detector. Information concerning the position of the optical filter 35 as a function oftime is l~ Lled to the data processing system, and used to permit inte,~,el~Lion ofthe fluorescence data. Thus, when the optical filter 35 is in a position that corresponds to the emission spectrum of the tag used to label sample I, the data processing system interprets the emission intensity as data for the sequence of sample 1, when the optical filter 35 is in a position that corresponds to the emission spectrum of the tag used to label sample 2, the data processing system interprets the emission intensity as data for the sequence of sample 2 and so on for as many dirrel ~nl tags as are used.
Finally, in the case where the properties of the selected tags are of the type shown in Fig. 2C, both optical filter 32 and optical 35 are adjustable in synchro~liG~lion to control the excitation and emission wavelengths being monitored. Information concerning the position of the optical filters 32 and 35 as a function oftime is transmitted to the data processing system, and used to permit interpretation ofthe fluorescence data. Thus, when optical filter 32 is in a position that corresponds to excitation spectrum Exl in Fig 2C, and optical filter 35 is in a position that l,anslllils the light of the wavelength of emission spectrum Eml, the data processing system interprets the emission intensity as data for the sequence of the sample . . .

labeled with tag 1. When the optica} filter 35 is in a position to l~ slll;l light ofthe wavelength of emission spectrum Em2, on the other hand, the data processing system interprets the emission intensity as data for the sequence of the of sample labeled with tag 2.
When absorption of light rather than emission of light is monitoredl the apparatus still 5 has subst~ntially the structures shown in Fig. 3. In this case, however, any optical filters employed before and after the separation matrix are selected to pass light of the same wavelength, corresponding to an absorption of the selected spectroscopically-detectable label.
The light source 31 in the appal ~tus of the invention may be a single light source which is moved across the gel to irradiate each detection zone individually, for example as described in US Patent No. 5,207,880 which is incorporated herein by reference. The light source 31 may also be a singe light source which is split into multiple beamlets, for example using optical fibers or through the use of a beam splitter such as a spot array generation grating to each of the detection sites as described generally in US Patent Application Serial No. 0~/353,932 and PCT Patent Application No. PCT/US95/15951 which are incorporated herein by reference.
The light source 31 may also be multiple individual light sources, each of which irradiates a subset of one or more of the detection sites within the separation matrix.
While optical filter 32 described above can be used effectively to provide selection of excitation wavelength, it will be understood that other approaches to providing di~relenl excitation wavelengths can be used as well. For example a plurality of lasers of different wavelengths could be used, with the light from each directed in turn to the detection site. For detection of product oligonucleotide fragments in multiple lanes of an electrophoresis gel, one set of lasers might be used (one for each necessary wavelength) ~,vith light from each of the lasers being conducted by optical fibers or through the use of a beam splitter such as a spot array generation grating to each of the detection sites as described generally in US Patent Application Serial No. 08/353,932 and PCT Patent Application No. PCT/US95/ 15951.
Optical switches, operatively connected to the data processing system, are used to select which of the excitation wavelengths is striking the detection zone at any given time, in the same manner as a rotating optical filter could do. A detector assembly which scans across the lanes of an electrophoresis gel could also be used, for example as described in US Patents Nos. 5,360,523 and 5,100,529, which are incorporated herein by reference, although the time .

CA 022~1809 1998-10-13 WO 97/40184 PCT/CA97/002Sl required for such a device to scan all the lanes of a gel may be a limiting factor in applications with short total migration times.
It will also be appreciated that multiple optical filters mounted on individual detectors could be used in place ofthe adjustable optical filter 35 and the single detector 36 shown in 5 Fig. 3. Similarly several detectors with adjustable optical filters might also be used.
Other optical components which separate light by wavelength may also be used in place of optical filter 3 5 . Thus, for example, a diffiaction grating or prism which spatially separates light of differing wavelength may be employed. In this case, radiation from the dirre~ t;"l fluorophores will be distributed in space, and can be detected by dedicated detectors 10 such as photodiodes, CCD elements (linear or X-Y). Similarly, distinct optical filters for each wavelength can be employed in combination with a multiplicity of detectors. Optical filters which are transmissive at the emission wavelength of one fluorophore and reflective at the emission wavelength of a second fluorophore can also be used to direct emitted light to separate detectors depending on wavelength.
The detectors used in the apparatus of the invention may be photomultipliers, photodiodes or a CCD. As noted above, the apparatus can be configured with one or more detectors for each detection site. The apparatus can also be configured such that one detector overlaps with several detection sites if the excitation light is directed to the detection sites one at a time. In addition, the apparatus can use a single detector (or a small array of detectors) 20 which is sc~nned across the gel during detection.
For determining the sequence of a selected region of DNA using the method of theinvention, a kit may be form~ ted. Such a kit comprises, in packaged combination, a plurality of containers, each cont~ining a reagent for the sequencing of the selected region of DNA. The reagent in each container has a reactive portion, which is involved in the 25 sequencing reaction, and a fluorescent label portion. The label portions of the reagents in each container are dilrel e"l and (li~tingnich~ble one from the other on the basis of the excitation or emission spectra thereof.
In a pr~re, I ed embodiment, a kit in accordance with the invention comprises a plurality of primers for sequencing the selected region, each of the primers having a di~l enl and 30 rli.ctinvllich~hle fluorescent label. Thus, the reactive portions ofthe reagents in this case are the oligonucleotide primer to which the labels are ~tt~ched. The reactive portions may be .J ~ + 1'~ J~ i I I L . . ~ ' J - ._ l d - l L - ' - - CA 02251809 1998-10-13 -dif~ercnt f~om olle ~other, but are preferably the same~ Such a kit may also include additional reag~Dts for seql~cin~ including polymer~se enzymes, dideoxynucleotides and buffers.
Alternati~ely, the kit may contain one primer f~r the selected regicn ~nd a plurality of containers of chain-termin~ting nucleotides, each labeled wllh a different and distinguich~ble S fluarescent label. In this case, the reactive portion of the re~gent is ~he chain t~-min~in~
nuclcotide which can be illCOlt)ur~t~ in place of a no~al nucl~otide during thc sequencir~g reaction. The Icit may include reagent~ havin~ just one type of c~ain-t~rminatin~ nucleotido, for example ddA with a plurality ~f distinct flll~rescent labc]s, o~ it may include reag~nts llaving two or nlore types of chain-tenninating nucleotides. each with ~ plurality of distincl 10 labels. Such a kit may also incl~de add-~ion~l rea~ents for SeQ~Ien~ing7 inclllding polyrnera~e enzysnes and ~uf~ers, as well as ~ t ~n31 ch~in-~ermin~Ling nucieolidcs (single-labeled) for those not pr~vided as part of a Ir;ulti-label re3gent set.
Forpracticing IJle method shown in ~ig. IB~ a suitable kit in aecolda~lce with the invenrion inciude,~ at lcast one contain~r eonlaining a mixture of a pluraiity of seque~cing 15 primers, one for each ,~,ene r~gion to be evaluated. The pl~rality of seq~encing primers e~ch comprise a re~ctive portion whcll hybridizes with DNA in the sarnple and a label portion, the la~el por~ions l~f ~he reagent~ ~eing different and distinguishable ane fr~m the Gther~
Pref~rably, the detectable labels are spectr~scopically-detecta~le tags, distin~uish~ble one ~om t~e oth~r by their emic.~ n or excitati~n spectra.

~ ,~o~n St~~E~

,

Claims

-11-

1. A method for real time evaluation of the nucleic acid sequence of a target nucleic sequence within a plurality of samples comprising the steps of (a) obtaining a first aliquot of each sample;
(b) combining the first aliquot of each sample with a sequencing reaction mixture containing a polymerase enzyme, a sequencing primer for hybridizing with the target nucleic acid sequence, nucleotide feedstocks and a first dideoxynucleotide to form a first plurality of mixtures of product oligonucleotide fragments, one for each sample, wherein the product oligonucleotide fragments formed from each first aliquot are labeled with different spectroscopically-detectable tags, said different spectroscopically-detectable tags being distinguishable one from the other on the basis of their excitation of fluorescent emission spectra;
(c) combining the first plurality of mixtures of oligonucleotide products to form a first combined mixture;
(d) loading the first combined mixture onto a separation matrix at a first loading site;
(e) applying an electric field to cause the oligonucleotide products in the first combined mixture to migrate within the separation matrix; and (f) detecting the oligonucleotide products having the different spectroscopically-detectable tags as they migrate within the separation matrix.

2. A method for real-time evaluation of the sequence of a target nucleic acid polymer in a plurality of samples comprising the steps of (a) obtaining four aliquots of each sample;
(b) combining the aliquots of each sample with four sequencing reaction mixtures, each sequencing reaction mixture containing a polymerase enzyme, a primer for hybridizing with the target nucleic acid, nucleotide feedstocks and a different dideoxynucleotide to form an A-mixture, a G-mixture, a T-mixture and a C-mixture for each sample containing product oligonucleotide fragments of varying lengths, wherein the product oligonucleotide fragments are labeled with spectroscopically-detectable tags, and the spectroscopically-detectable tags used for each sample are distinguishable one from the other on the basis of their excitation or fluorescent emission spectra;
(c) combining the A-mixtures, the G-mixtures, the T-mixtures and the C-mixtures for each sample to form a combined A-mixture, a combined G-mixture, a combined T-mixture and a combined C-mixture;
(d) loading the combined A-mixture, the combined G-mixture, the combined T-mixture and the combined C-mixture onto a separation matrix at separate loading sites;
(e) applying an electric field to cause the product oligonucleotide fragments in the combined mixtures to migrate within the separation matrix; and (f) detecting the product oligonucleotide fragments having the different spectroscopically-dotectable tags as they migrate within the separation matrix.

3. A method for evaluation of the sequence of a plurality of gene regions within a sample comprising the steps of:
(a) combining at least a first aliquot of the sample with a sequencing reaction mixture containing a polymerase enzyme, a plurality of sequencing primer species, one sequencing primer species for each gene region, nucleotide feedstocks and a first dideoxynucleotide to form a first mixture of product oligonucleotide fragments, wherein each of the sequencing primer species is labeled with a different detectable label, said different detectable labels being distinguishable one from the other by a detection system;
(b) separating the first mixture of product oligonucleotide fragments based upon the size of the fragments;
(c) detecting emissions from the separated oligonucleotide fragments for each different detectable label; and (d) evaluating the sequence of each gene region based upon the oligonucleotide fragments detected.

4. The method according to claim 1, further comprising the steps (i) obtaining a second aliquot of each sample;

(ii) combining the second aliquot of each sample with a sequencing reaction mixture containing a polymerase enzyme, a primer for hybridizing with the sample, nucleotide feedstocks and a second dideoxynucleotide different from the first dideoxynucleotide to form a second plurality of mixtures of product oligonucleotide fragments, one for each sample, wherein the product oligonucleotide fragments formed form each second aliquot are labeled with different spectroscopically-detecable tags, said different spectroscopically-detectable tags being distinguishable one from the other on the basis of their excitation or emission spectra;
(iii) combining the second plurality of mixtures of oligonucleotide products to form a second combined mixture;
(iv) loading the second combined mixture onto a separation matrix at a second loading site, distinct from the first loading site;
(v) applying an electric field to cause the oligonucleotide products to migrate within the separation matrix; and (vi) detecting the oligonucleotide products having the different spectroscopically-detectable tags as they migrate within the separation matrix.

5. The method according to claim 4, wherein the first and second aliquots are combined with a sequencing reaction concurrently, and wherein the first and second combined mixtures are loaded onto different lanes of the same gel.

6. The method according to claim 5, wherein the same prime and spectroscopically-detectable tag are combined with the first and second aliquots of each sample.

7. The method according to claim 1 or 2, wherein first aliquots from at least three samples are combined to form the first combined mixture.

8. The method according to claim 1 or 2, wherein first aliquots from mare than four samples are combined to form the first combined mixture.

9. The method according to claim 3, wherein the spectroscopically-detectable tagis a fluorescent tag.

10. An apparatus for evaluating the sequence of a nucleic acid polymer by separation of a reaction mixture containing oligonucleotide fragments in a separation matrix disposed within the apparatus comprising (a) means for providing excitation energy to a detection site within the separation matrix;
(b) means for detecting light emitted from fluorescently-labeled oligonucleotide fragments located within the detection zone;
(c) configuration control means, operatively connected to the means for providing excitation energy and the means for detecting to provide combinations of excitation wavelength and detection wavelength specific for a plurality of different fluorescently-labeled oligonucleotide fragments; and (d) data processing means, operatively connected to the configuration control means and the means for detecting for receiving a signal from the means for detecting and assigning that signal to a data stream associated with a particular sample based upon the combination of excitation wavelength and detection wavelength set by the configuration control means.

11. A kit for determining the sequence of a selected region of DNA in accordance with the method of any of claims 1 to 9 comprising, in packaged combination, at least of first set of a plurality of containers, each container containing a reagent species for the sequencing of the selected region of DNA, wherein the reagent species in each container comprises a reactive portion and a label portion and wherein the label portions of the reagents are different and distinguishable one from the other.

12. A kit for evaluating the sequence of a plurality of gene regions within a sample in accordance with claim 3, comprising, in packaged combination, at least a first container containing a mixture of a plurality of sequencing primers, one for each gene region to be evaluated, wherein the plurality of sequencing primers each comprise a reactive portion which hybridizes with DNA

in the sample and a label portion and wherein the label portion of the reagents are different and distinguishable one from the other.

13. The kit according to claim 11 or 12, wherein the detectable labels are spectroscopically-detectable tags, distinguishable on the basis of the excitation or emission spectra thereof.

14. The kit according to any of claims 11 through 13, wherein the reactive portion of each reagent species in the first set of containers is all oligonucleotide primer for use in sequencing the selected region of DNA.

15. The kit according to claim 14, wherein the reactive portions of each reagent species in the first set of containers are the same as one another.

16. The kit according to claim 11, wherein the reactive portion of each reagent species in the first set of containers is a dideoxynucleotide and wherein the active portions of each reagent species in the first set of containers are the same as one another.

17. The kit according to any of claims 11 or 13 through 16, further comprising a second set of containers, each containing a reagent species for the sequencing of the selected region of DNA, wherein the reagent species in each container of the second set comprises a dideoxynucleotide reactive portion different from the reactive portion of the reagent species in the first set of containers, and a label portion and wherein the label portions of the reagents in the second set of containers are different and distinguishable one from the other on the basis of the excitation or emission spectra thereof.