CA1258611A

CA1258611A - Method of dna sequencing

Info

Publication number: CA1258611A
Application number: CA000472103A
Authority: CA
Inventors: Lloyd M. Smith; Tim J. Hunkapiller; Leroy E. Hood; Michael W. Hunkapiller
Original assignee: California Institute of Technology
Current assignee: California Institute of Technology
Priority date: 1984-01-16
Filing date: 1985-01-15
Publication date: 1989-08-22
Also published as: GB2155176A; DE3501306C2; DE3501306A1; FR2558262B1; SE8500201D0; SE456348B; SE8500201L; GB2155176B; GB8500960D0; FR2558262A1

Abstract

ABSTRACT OF THE DISCLOSURE

A process for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations wherein a set of four chromophores or fluorophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel.
A system for the analysis of DNA fragments comprising:
a source of chromophore or fluorescent tagged DNA fragments, a zone for contacting an electrophoresis gel; means for intro-ducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

Description

1 ¦ BACKGROUND OF T~IE INVENTION

2 1

3¦ The development of reliable methods for sequence analysis

4 ¦ of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) has been

5 ¦ one of the keys to the success of recombinant DNA and genetic en-

6 ¦ gineering. When used with the other techniques of modern molecular

7 ¦ biology, nucleic acid sequencing allows dissection and analysis of

8 ¦ animal, plant, and viral genomes into discrete genes with defined

9 ¦ chemical structure. Since the function of a biological molecule is determined by its structure, defining the structure of a ~ene is 11 ¦ crucial to the eventual manipulation of this basic unit of heredi-12 ¦ tary information in useful ways. Once genes can be isolated and 13 ¦ characterized, they can be modified to produce desired changes in 14 ¦ their structure that allow the production of gene products--15 ¦ proteins--with different properties than those possessed by -the 16 ¦ original proteins. Microorganisms into which the natural or 17 ¦ synthetic genes are placed can be used as chemical "facto~ies" to 18 ¦ produce large amounts of scarce human proteins such as interferon, 19 ¦ growth hormone, and insulin. Plants can be given the genetic 20 ¦ information to allow them to survive harsh environmental conditions 21 ¦ or produce their own fertilizer.
22 ¦ The development of modern nucleic acid sequencing methods 23 ¦ involved parallel developments in a variety of techniques. One 24 ¦ was the emergence of simple and reliable methods for cloning small to medium-sized strands of DNA into bacterial plasmids, bacterio-26 phages, and small animal viruses. This allowed the production of 27 pure DNA in sufficient quantities to allow its chemical analysis.
28 Another was the near perfection of gel electrophoretic methods ~, ~5~

1 for -the high resolution separation of oligonucleo-tides on -the basis 2 oE their size. The key conceptual development, however, was the 3 introduction of methods of genera-ting size-nested sets of fragments 4 cloned, purified DNA that contain, in their collection of lengths, the information necessary to define the sequence of the nucleo-6 tides comprising the parent DNA molecules. Two distinct methods 7 of generating these fragment sets are in widespread use, one 8 developed by Sanger; Sanger, F., Nicklen, S. and Coulson, A. R.
9 Proc. Natl. Acad. Sci. USA 74, 5463 (1977) and Smith, A. J. ~1.
Methods in Enzymology 65, 56-530 (1980); the other by Maxam and 11 Gilbert; Maxam, A. M. and Gilbert, W. Methods in Enzymology 65, 12 499--559 (1980).
13 The method developed by Sanger is referred to as the dideoxy 14 chain termination method. In the most commonly used variation of this method, a DNA segment is cloned into a single-stranded DNA
16 phage such as M13. These phage DNAs can serve as templates for 17 the primed synthesis of the complementary strand by the Klenow 18 fragment of DNA polymerase I. The primer is either a synthetic 19 oligonucleotide or a restriction fragment isolated from the parental recombinant DNA that hybridizes specifically to a region 21 of the M13 vector near the 3' end of the cloned insert. In each 22 of four sequencing reactions, the primed synthesis is carried 23 out in the presence of enough of the dideoxy analog of one of 24 the four possible deoxynucleotides so that the growing chains are randomly terminated by the incorporation of these "dead-end"
26 nucleo-tides. The relative concentration of dideoxy to deoxy forms 27 is adjusted to give a spread of termination events corresponding 28 to all the possible chain lengths that can be resolved by gel ~,, 12S~

1 electrophoresis. The products from each of the four primed synthe-2 sis reactions are then separated on individual tracks of polyacryl-3 amide gels by the electrophoresis. Radioactive tags incorporated 4 in the growincJ chains are used to develop an autoradiogram image of the pattern of the DNA in each electrophoresis track. The 6 sequence of the deoxynucleotides in the cloned DNA is determined 7 from an examination of the pattern of bands in the four lanes.
8 The method developed by Maxam and Gilbert uses chemical 9 treatment of purified DNA to generate size-nested sets of DNA
fragments analogous to those`produced~by the Sanger method.
11 Single or double-stranded DNA, labeled with radioactive phosphate 12 at either the 3' or 5' end, can be sequenced by this procedure.
13 In four sets of reactions, cleavage is induced at one or two of 14 the four nucleotide bases by chemical treatment. Cleavage in-volves a three-stage process: modification of the base, removal 16 of the modified base from its sugar, and strand scission at that 17 sugar. Reaction conditions are adjusted so that the majority of 18 end-labeled fragments generated are in the size range (typically 19 1 to 400 nucleotides) that can be resolved by gel electrophoresis.
The electrophoresis, autoradiography, and pattern analysis are 21 carried out essentially as is done for the Sanger method.
22 (Although the chemical fragmentation necessarily generates two 23 pieces of DNA each time it occurs, only the piece containing the 24 end label is detected on the autoradiogram~) Both of these DNA sequencing methods are in widespread use, 26 and each has several variations. For each, the length of sequence 27 that can be obtained from a single set of reactions is limited pri-28 marily by the resolution of the polyacrylamide gels used for ~:~

1 elec~rophoresis. Typically, 200 -to ~00 bases can be read from a single set of gel tracks. ~lthough successful, both methods have 3 ¦ serious drawbacks, problems associated primarily with -the electro-41 phoresis procedure. One problem is the requirement of the use of 5 ¦ radiolabel as a -tag for location of the DNA bands in the gels.
6 ¦ One has to contend with the short half-life of phosphorus-32, 7 ¦ and hence the instability of -the radiolabeling reagents, and with 8 ¦ the problems of radioactive disposal and handling. More impor-9 1 tantly, the nature of autoradiographY (the film image of a radio-active gel band is broader than the band itself) and the comparison 11 ¦ of band positions between four different gel -tracks (which may or 12 1 may not behave uniformly in terms of band mobilities) can limit 13 ¦-the observed resolution of bands and hence the length of sequence 14 that can be read from the gels. In addition, the track-to-track 15 ¦irregularities make automated scanning of the autoradiograms 16 ¦difficult--the human eye can presently compensate for these ir-17 ¦regularities much better than computers can. This need for 18 ¦manual "reading" of the autoradiograms is time-consuming, tedious 19 and error-prone. Moreover, one cannot read the gel patterns while the electrophoresis is actually being performed, so as to be able 21 to terminate the electrophoresis once resolution becomes insuf-22 ficient to separa-te adjoining bands, but must terminate the electro-23 phoresis at some standardized time and wait for the au-toradiogram 24 to be developed before -the sequence reading can begin.
The present invention addresses these and other problems as-26 sociated wi.th the electrophoresis step in the DNA se~uencing pro-27 cedures and is believed to represent a significant advance in the 28 art.
~,~

~ 68299-79 SUMMARY OF THE INVENTION
Brie~ly, this invention comprises a novel process for the electrophoretic analysis of DNA fragments produced in DNA
sequencing operations wherein at least one chromophore or fluoro-phore are used to tag the DNA fragments produced by the sequencing chemistry and permit -the detection and characterization of the fragments as they are resolved by electrophoresis through a gel. The detection employs an absorption or fluorescent photometer capable of monitoring the tagged bands as they are moving through the gel.
This invention also includes a novel system for the electrophoretic analysis of DNA fragements produced in DNA
sequencing operations comprising:
a source of chromophore or fluorescent tagged DNA
fragments from sequencing operatlons, a zone for containing an eleckrophoresis gel, means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring or detecting said tagged DNA fragments as they move through and are separated by said gel.
It is an object of this invention ko provide a novel process for the sequence analysis of DNA.
It is another object of our invention to provide a novel system for the analysis of DNA fragements.
More particularly, it is an object of this invention to provide an improved process for the sequence analysis of DNA.

~S8~ 68299-79 These and other objec-ts and advantages of this invention will be apparent from the detailed description which ~ollows taken in conjunction with the accompan~ing drawings.

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¦ DET~II,I~ DCSCI~IP'IIO~ 0~ IF INVENTI~N

3~ Turning to the drawings:
4 ¦ ~igure l is an illustra-tion of one means of end-labeling a 51 DNA fragment with a fluorescent tag. Pst. I and T4 DNA ligase are 6 1 enzymes commonly used in recombinant DNA research.
7 ¦ Figure 2 is a block diagram of automated DNA sequencer, gel 8 ¦ electrophoretic system.
9 ¦ Figure 3 is a schematic diagram of an optical confirguration

10 ¦ in the detector unit. P, lamp source; Ll, objective lens; L2, col-

11 ¦ limating lens; Fl, UV blocking fllter; F2, heat blocking filter;

12 ¦ F3, band pass excitation filter; F4, long pass emission filter; DM,

13 ¦ dichroic mirror; C, polyacrylamide gel; PMT, photomultiplier tube.

14 1 Figure 4 is a schematic diagram of another optical configur-

15 ¦ ation in the detector unit. Fl to F4 are bandpass filters centered

16 ¦ at the emission maximum of the different dyes. Pl to P4 are photo-

17 ¦ multiplier tubes. The excitation light is of a wavelength such

18 that it s not transmitted through any of the filters Fl to F4.

19 Figure 5 is a comparison of the type of data produced by DNA
sequencing of the sequence shown in Eigure l.
21 Figure 6 is a block diagram of a preferred DNA sequencer 22 according to this invention.
23 Design of tagged primers for use with Sanger method. In 24 the previous rnethods of DNA sequencing, including -those based on the Sanger dideoxy chain termination method, a single radioactive 26 label, phosphorus-32, is used to identify all bands on the gels.
27 This necessitates that the fragment sets produced in the four 28 synthesis reactions be run on separate gel tracks and leads to ~S~36~
the problern associated with comparing band mobilities in the dif~erent -tracks. ~his problem is overcome in the present invention by the use of a set of four chromophores or fluorophores with different absorption or fluorescent maxima, respectively.
Each of these tags is coupled chemically to the primer used to initiate the synthesis of the fragment strands. In turn, each tagged primer is then paired with one of the dideoxynucleotides and used in the primed synthesis reaction with the Klenow fragment of DNA polymerase.
The primers must have the following characteristics.
1) They must have a free 3'hydroxyl group to allow chain extension by the polymerase. 2) They must be complementary to a unique region 3' of the cloned insert. 33 They must be sufficiently long to hybridize to form a uniquel stable duplex.
4) The chromophore or fluorophore must not interfere with the hybridization or prevent 3'-end extension by the polymerase.
Conditions 1, 2 and 3 above are satisfied by several synthetic oligonucleotide primers which are in general use for Sanger-type sequencing utilizing M13 vectors. One such primer is the 12 mer 5'TCA CGA CGT TGT 3' where, A, C, G and T represent the four different nucleoside components of DNA; A, adenosine; C, cytosine; G, guanosine; T, thymidine.
The strategy used for the coupling of the chromophoric or fluorophoric tags is to introduce an aliphatic amino group at the 5' terminus as the last addition in the synthesis of the oligo-1 .~;251~6~

1~ nucleotide primer. This reactive amino group may then readily 2 ¦ be coupled with a wide variety or amino reac-tive chromophores 3 ¦ and/or fluorophores. ~his approach aids compatibility of the 4 ¦ labeled primers with condition 4 above.
5 ¦ The four dyes used must have high extinction coefficients 6 ¦ and/or reasonably high quantum yields for fluorescence. They must 7 ¦ have well resolved absorption maxima and/or emission masima. A
8 ¦ representative set of four such amino reactive dyes are: fluor-9 ¦ escein isothiocyanage (FITC, ~max = 495~ ~max = 520~ 495 8 x 10 ), eosin isothiocyanate (EITC, ~Ex = 522, ~ = 543, 11 ¦ 522 ~ 8 x 104), tetramethyl rhodamine isothiocyanate (TMRITC, 12 ¦ ~EXX = 550, AmmaX = 578, 550 ~ 4 x 104), and substituted 13 ¦ rhodamine isothiocyanate (XRITC, ~ = 580, ~ = 604, 580 14 ¦ ~ 8 x 104) where ~ represents the wavelength in nanometers, 15 ¦ EX is excitation, Em is emission, max is maximum, and ~ is the 16 ¦ molar extinction coefficient.
17 ¦ These dyes have been attached to the M13 primer and the 18 ¦ conjugates electrophoresed on a 20% polyacrylamide gel. The 19 labeled primers are visible by both their absorption and their fluorescence in the gel. All four labeled primers have identical 21 electrophoretic mobilities. The dye conjugated primers retain 22 their ability to specifically hybridize to DNA, as demonstrated 23 by their ability to replace the underivitized oligonucleotide 24 normally used in the sequencing reactions.
End labeling of DNA for use with Maxam/Gilbert method. In _ _ 26 the Maxam/Gilbert method of DNA sequencing, the end of the piece 27 of DNA whose sequence is to be determined must be labeled. This 28 is conventionally done enzymatically using radioactive nucleo-1 ~5~
1 j sides. In order to use the Maxam/Gilbert method in conjunction 2 ¦ with the dye detection scheme described in this invention, -the 3 ¦ DNA piece mus-~ be labeled with dyes~ One manner in which this 4 ¦ may be accomplished is shown in Figure 1. Certain restriction 5 ¦ endonucleases generate what is known as a 3' overhang as the pro-6 duct of DNA cleavage. These enzymes generate a "sticky end," a 7 ¦ short stretch of single stranded DNA at the end of a piece of 8 ¦ double stranded DNA. This region will anneal with a complementary 9 ¦ stretch of DNA, which may be covalently joined to the duplex DNA
10 ¦ with the enzyme ligase. In this manrier one of the strands is 11 ¦ covalently linked to a detectable moiety~ This moiety may be a 12 ¦ dye, an amino group or a protected amino group (which could be 13 ¦ deprotected and reacted with dye subsequent to the chemical 14 ¦ reactions).
15 I Sequencing reactions. The dideoxy sequencing reactions are 16 1 performed in the standard fashion Smith, A.J.H., Methods in En2y-17 ¦ mology 65, 56--580 (1980), except -that the scale may be increased 18 ¦ if necessary to provide an adequate signal intensity in each band 19 ¦ for detection. The reactions are done using a different color

20 ¦ primer for each different reaction, e.g., FITC for the "A" reaction ,

21 ¦ EITC for the "C" reaction, TMRITC for the "G" reaction, and XRITC

22 ¦ for the "T" reaction. No radiolabeled nucleoside triphosphate

23 ¦ need be included in the sequencing reaction.

24 ¦ The Maxam/Gilbert sequencing reactions are perEormed in the

25 ¦ usual manner, Gill, S. F. Aldrichimica Acta 16(3), 59-61 (1983),

26 ¦ except that the end label is either one or four colored dyes, or

27 ¦ a free or protected amino group which may be reacted with dye

28 1 subsequently.
I

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1 j Gel electrophoresis. ~liquots of the sequencing reactions 2 ¦ are combined and loaded onto a 596 polyacrylamide column 10 shown 3 ¦ in Figure 2 f~om -the upper reservoir 12. The relative amounts of 4 ¦ the four different reactions in the mixture are ernpirically ad-5 ¦ justed to give approximately the same fluorescence or absorptive 6 ¦ signal intensity from each of the dye DNA conjugates. This per-7 ¦ mits compensation for differences in dye extinction coefficients, 8 ¦ dye fluorescence quantum yields, detector sensitivities and so on.
9 1 A high voltage is placed across the column lO so as to electro-phorese the labeled DNA fragments through the gel. The labeled 11 ~ DNA segments differing in length by a single nucleotide are 12 ¦ separated by electrophoresis in this gel matrix. At or near the 13 1 bottom of the gel column lO, the bands of DNA are resolved from 14 ¦ one another and pass through the detector 14 (more fully described 15 1 in the next section). The detector 14 detects the fluorescent or 16 ¦ chromophoric bands of DNA in the gel and determines their color, 17 ¦ and therefore to which nucleotide they correspond. This informa-18 ¦ tion yields the DNA sequence.
19 ¦ Detection. There are many different ways in which the 20 ¦ tagged molecules which have been separated by length using poly-21 ¦ acrylamide gel electrophoresis may be detected. Four illustra-22 1 tive modes are described below. These are i) detection of the 23¦ fluorescence excited by light of different wavelengths for the 24 ¦ different dyes, ii) detection of fluorescence excited by light o~
25 ¦ the same wavelength for the different dyes, iii) elution of the 26 1 molecules from the gel and detection by chemiluminescence, and iv) 27 ¦ detection by -the absorp-tion of light by the molecules. In modes i) 28 ¦ and ii) the fluorescence de-tector should fulfill the following I

~'~5~

1 requiremen~s. a) The excitation light beam should not have a 2 ~ height substantially greater than the height of a band. This is 3 ¦ normally in the range of 0.1 to 0.5 mm. I~he use of such a narrow L~ ¦ excitation beam allows -the attainment of maximum resolution of 5 ¦ bands. b) The excitation wavelength can be varied to ma-tch the 6 ¦ absorption maxima of each of the different clyes or can be a single 7 ¦ narrow, high in-tensity light band that excites all four fluoro-8 ¦ phores and does not overlap with any of the fluorescence emission.
9 ¦ c) The optical configuration should minimize the flux of scatterec 10 ¦ and reflected excitation light to thé photodetector 14. The optical 11 ¦ filters to block out scattered and reflected excitation light are 12 ¦ varied as the excitation wavelength is varied. d) The photode-13 ¦ tector14 should have a fairly low noise level and a good spectral 14 ¦ response and quantum efficiency throughou-t the range of the emis-15 ¦ sion of the dyes (500 to 600 nm for the dyes listed above). e) 16 ¦ The optical system for collection of the emitted fluorescence 17 ¦ should have a high numerical aperture. This maximizes the fluor-18 ¦ escence signal. Furthermore, the depth of field of the collection 19 optics should include the entire width of the column matrix.
Two illustrative fluorescence detection systems are dia-21 grammed in Figures 3 and 4. The system in Figure 3 is compatible 22 with either single wavelength excitation or multi wavelength exci-23 tation. For single wavelength excitation, the filter F4is one of fou 24 band pass filters centered at the peak emission wavelength of each of the dyes. This filter is switched every few seconds to allow 26 continual monitoring of each of the four fluorophores. For multi 27 wavelength excitation, the optical elements F3 (excitation filter), 28 DM (dichroic mirror), and F4 (barrier filter) are switched to-1 getner. In this manner both -the excita-tion light and the observed 2 ¦ emission light are varied. The system in Figure A is a good 3 ¦ arrangement for the case of single waveleng-th excitation. This 4 ¦ sys~em has the advantage that no moving par-ts are required, and 5 ¦ fluorescence from all four of the dyes may be simultaneously and 6 ¦ continuously monitored. A third approach (iii above) to detection 7 ¦ is to elute -the labeled molecules a-t the bottom of the gel, com-8 ¦ bine them with an agent for excitation of chemiluminescence such as 9 ¦ 1,2 dioxetane dione, Gill, S. K. Aldrichimica Acta 16(3), 59-61 (1983); Mellbin, G. J. Liq. Chrom. 6~9), 1603-1616 (1983), and 11 ¦ flow the mixture directly into a detector which can measure the 12 1 emitted light at four separate wavelengths (this detector is similar 13 ~ to that shown in Figure 4, but without a need for an excitation 14 ¦ light source). The background signal in chemiluminescence is 15 ¦ much lower than in fluorescence, resulting in higher signal to 16 ¦ noise ratios and increased sensitivity. Finally, the measuxement 17 may be made by measurements of light absorption (iv above). In 18 this case, a light beam of variable wavelength is passed through 19 the gel, and the decrease in the beam intensity due to absorption 20 of light by the labeled molecules is measured. By measuring the 21 absorption of light at the different wavelengths corresponding 22 to the absorption maximum of the four dyes, it is possible to 23 determine which dye molecule is in the light path. A disadvantage ~4 of this type of measurement is that absorption measurements are 25 inherently less sensitive than fluorescence measurements.
26 The above-described detection system is interfaced to a com-27 puter 16. In each time interval examined, the computer 16 receives 28 a signal proportional to the measured signal intensity at that .,~

6~

l ~ time Eor each of the four colorecl tags. This informa-tion tells 2 which nucleotide terminates the DNA fragment of the particular 3 len~th in the observation window at that time. The temporal se-L~ quence of colored bands gives the DNA sequence. In Figure 5 is shown the type of data obtained by conventional methods, as well 6 as the type of data obtained by the improvements described in this 7 invention.
8 The following Example is presented solely to illustrate 9 the invention.
. ,.

13 Figure ~ shows a block diagram oE a DNA sequenator for use 14 with one dye at a time. The beam (4880 A) from an argon ion laser lO0 is passed into the polyacrylamide gel tube (sample) 102 by 16 means of a beamsteerer 104. Fluorescence exited by the beam is 17 collected using a low f-number lens lO~, passed through an appro-18 priate set oE optical filters 108 and llO to eliminate scattered 19 excitation light and detected using a photomultiplier tube ( PMT ) 112. The signal is readily detected on a strip chart recorder.
21 DNA sequencing reactions are carried out utilizing a fluorescein 22 labeled oligonucleotude primer. The peaks on the chart correspond 23 to fragments to fluorescein labeled DNA oE varying lengths synthe-24 sized in the sequencing reactions and separated in the gel tube by electrophoresis. Each peak contains the order oE lO to lO
26 moles of fluorescein, which is approximately equal to the amount 27 of DNA obtained per band in an equivalent sequencing gel utilizing 28 radioisotope detection. This proves that the fluorescent tag is ~5~

1 not removed or clegraded from the oligonucleotide primer in the 2 sequencing reac-tions. It also demonstra-tes that the detection 3 sensitivity is qu.ite adequate to perform DNA sequence analysis 4 by this means.
Havin~ fully described the invention it is intended that ¦ be llmited only by the l~wful sc~pe of the appended claims

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A novel system for the electrophoretic analysis of DNA
fragments produced in DNA sequencing operations comprising:
a source of chromophore or fluorescent tagged DNA
fragments from sequencing operations, a zone for containing an electrophoresis gel, means for introducing said tagged DNA fragements to said zone; and photometric means for monitoring said tagged DNA
fragments as they move through said gel.

2. The novel system of claim 1 wherein the photometric means is an absorption photometer.

3. The novel system of claim 1 wherein the photometric means is an fluorescent photometer.

4. The novel system of claim 1 wherein the DNA fragements are labeled with an amino group which is coupled to a dye molecule.

5. The novel system of claim 1 wherein a set of four chromophores or fluorophores are present to tag said DNA
fragments from sequencing operations.

6. A novel system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising:
a source of chromophore or fluorescent tagged DNA
fragments from sequencing operations;

a zone containing an electrophoresis gel;
means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA
fragments as they move through said gel.

7. The novel system of claim 6 wherein said source of tagged DNA fragments from sequencing operations is positioned at one end of said zone, and said detector is positioned in proximity to the opposite end of said zone.

8. The novel system of claim 6 wherein a set of four chromophores or fluorophores are present to tag said DNA
fragments from sequencing operations.

9. A process for the electrophoretic analysis of DNA
fragements produced in DNA sequencing operations which comprises providing tagged DNA fragements having at least one chromophore or fluorophore produced by the sequencing chemistry, and detecting said fragments as they are resolved by electrophoresis through a gel.

10. The method of DNA sequencing by the chain termination method according to claim 9 wherein a primer oligonucleotide labeled with a coloured tag is used.

11. The method of DNA sequencing by the chain termination method according to claim 9 wherein a primer oligonucleotide labeled with a fluorescent tag is used.

12. The method of DNA sequencing by chemical degradation method according to claim 9 wherein DNA molecules labeled with a coloured tag are used.

13. The method of DNA sequencing by chemical degradation method according to claim 9 wherein DNA molecules labeled with a fluorescent tag are used.

14. The method of DNA sequencing according to claim 9 wherein a set of four chromophores or fluorophores are used to tag said DNA fragments produced by the sequencing chemistry.

15. In the method of DNA sequencing by the chain termination method according to claim 9;
the improvement wherein the primer oligonucleotide used in each of the four sequencing reactions, A, C, G and T, has a different coloured tag attached to it, and wherein aliquots of the aforesaid sequencing reactions are combined and electro-phoresed together on polyacrylamide gel and detected after their separation on the gel.

16. In the method of DNA sequencing by the chain termination method according to claim 9;
the improvement wherein the primer oligonucleotide used in each of the four sequencing reactions, A, C, G and T, has a different fluorescent tag attached to it, and wherein aliquots of the aforesaid sequencing reactions are combined and electro-phoresed together on polyacrylamide gel and detected after their separation on the gel.

17. In the method of DNA sequencing by chemical degradation method according to claim 9;
the improvement wherein the DNA molecules are labeled with different coloured tags, and a different coloured DNA is used in each of the chemical modification reactions, and aliquots of the aforesaid sequencing reactions are combined and electro-phoresed together on a polyacrylamide gel and detected after their separation of the gel.

18. In the method of DNA sequencing by chemical degradation method according to claim 9;
the improvement wherein the DNA molecules are labeled with different fluorescent tags, and a different fluorescent DNA
is used in each of the chemical modification reactions, and aliquots of the aforesaid sequencing reactions are combined and electrophoresed together on a polyacrylamide gel and detected after their separation of the gel.

19. In the method of DNA sequencing by chemical degradation method according to claim 9;
the improvement wherein the DNA molecules are provided with an amino group, which is coupled to a dye molecule subsequent to the sequencing reactions.

20. In the method of DNA sequencing by chemical degradation method according to claim 9;
the improvement wherein the DNA molecules are provided with a protected amino group, which is deblocked and coupled to a dye molecule subsequent to the sequencing reactions.

21. In the method of claim 19, the further improvement wherein the products of each of the different sequencing reactions are coupled with a different colour dye, aliquots of the dye labeled reaction are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel.

22. In the method of claim 20, the further improvement wherein the products of each of the different sequencing reactions are coupled with a different colour dye, aliquots of the dye labeled reaction are combined and electrophoresed on a polyacrylamide gel and detected after their separation on the gel.