BACKGROUND OF THE INVENTION

Field of the Invention

This invention is in the field of sequencing nucleic acids.

Background of the Prior Art A major goal of the Human Genome Project is to determine the entire sequence of the human genome by the year 2005. Substantial improvements in DNA sequencing technologies will be required to achieve this ambitious goal. Two major issues important for achieving this goal are throughput of the sequencing step (bases per hour) and read length (bases). Higher throughput increases the productivity of the sequencing step, and longer read lengths reduce the pre- and post-sequencing processing required to produce completed sequence information.

Arguably, the preferred methods of sequencing DNA at the present are those derived from the protocol described by Sanger, Niklen, and Coulson. Sanger et al (F. Sanger, S. Niklen, and A.R Coulson, Proc NatL Acad. Scl 1977 74 5643) first developed one of the two basic methods of DNA sequencing; they described an enzymatic method of generating 4 sets of discrete DNA sequencing fragments using a primer and a DNA polymerase, with each subset of fragments labeled and terminated with one of four dideoxynucleoside triphosphates to correspond to an individual nucleoside base (adenosine A cytosine C, guanosine G, or thyrnidine T). The individual fragments were labeled with a radioisotope ("P, ³ⁱ S, etc.) which allowed sensitive detection of the fragments via autoradiography after separation of the individual sets of fragments according to size in individual lanes of an electrophoreuc slab gel. Because band mobilities can differ between adjacent lanes of an electrophoreuc gel, "reading" the DNA sequence from the order of migration of the fragments across four adjacent lanes requires substantial skill and practice. In addition, the

use of a radioisotope label for DNA detection makes this sequencing process inconvenient

Smith et al (U.S. 5,171,534; Nature 1986 321(12) 674-679) improved upon the original Sanger sequencing methodology by substituting four fluorescently-labeled dyes for the radioisotope label. The dyes were attached to the primer used in the enzymatic extension/te minauόn reactions and were color-coded such that after completion of the four extension/termination reactions to generate the set of 4 sequencing fragments corresponding to the individual nucleoside bases, the 4 sets of fragments could be combined and subsequently simultaneously separated and detected in real-time via fluorescence in a single electrophoreuc separation.

Prober and colleagues (Science 1987238336-341; U.S.5,332,666; U.S. 5,242,796; U.S.5,306,618) described another improvement of the Sanger primer extension/chain termination sequencing method; the improved system incorporates the four color-coded fluorescent detection labels onto the individual dideoxy-based chain terminators (designated T-526, C-519, A-512, G- 505, where the letter specifies the termination base and the number specifies the lλ of ma-rirnιιrn fluorescent emission). This improvement allows the four chain extei on/teππination reactions to be performed in a single reaction mixture which generates four sets of color-coded terminated fragments, each set associated with one of the individual nucleoside bases in the DNA.

The above sequencing methods all rely on the use of slab gel electrophoresis for the separation of the sequencing fragments according to size. Though effective, this separation method is slow and labor intensive, while requiring large quantities of gel preparation reagents, run buffers, sample, etc. It was recognized that these limitations could be overcome by capillary electrophoresis. Initial efforts to separate DNA sequencing fragments by capillary electrophoresis centered on the use of gel-filled capillaries, in which narrow-bore fused silica capillaries were filled with cross-linked polyacrylamide gels (e.g., Swerdlow, R. and R. Gesteland, Nucleic Acids Research 1990 18 1415-1419; Zagursky, R. J. and R. M. McCormick, BioTechniques 1990 9(1) 74-

79). Problems associated with production and use of such cross-linked, gel- filled capillaries, such as void formation in the gel matrix during polymerization and reproducibility and stability of such gels during extended periods of use, made this approach for separating sequencing fragments undesirable. Recently, however, the use of flowable, non-crosslinked sieving matrices termed "sieving buffers" has been demonstrated for separation of labeled terminated nucleic acid fragments. Such "buffers" are actually solutions of a polymer, which when used in sufficiently high concentration, form a sieving matrix due to overlap of the strands of the polymer in solution. Overlap of adjacent polymer strands in solution forms transient pores, tile size of which is determined by the chemical nature, the molecular weight, and the concentration of the polymer. Sized-based separations of numerous types of biomolecules, including single-stranded DNA (e.g. DNA sequencing fragments), double-stranded DNA (e.g. restriction fragments, PCR fragments), proteins, oligosaccharides, etc., have been demonstrated within the past 5 years.

With regard to separation of DNA sequencing fragments such as base or primer labeled terminated nucleic acid sequences by capillary electrophoresis, efforts have centered on extending the read length in a single capillary. For example Best et al (Analytical Chemistry 1994 66 4063-4067) demonstrated read lengths to 570 bases in a 35 cm capillary in a time period of -2 hours. Similarly, Grossman (U.S. 5,374,527) demonstrated a read length to 500 bases in the same time period. Karger and colleagues (Analytical Chemistry 1993 652851-2858; Analytical Chemistry 1993653219-3228) obtained a read length of 350 bases in just over one-half hour. Similarly, Huang et al (Analytical Chemistry 64 2149-2154) showed multiple (25) separations where read length extended to 300-350 bases in about 2 hours; the separation matrix utilized for their separations was not readily replaceable because of the high viscosity of the sieving buffer and the length (40 cm) of the capillaries used. Finally, Manabe et al (Analytical Chemistry 1994 66 4243-4252) achieved resolution out to 520 bases in about 10 hours in a 55-cm long capillary. For approaches which utilize only a few capillaries in parallel and each capillary

separates the fragments of a different sample, extension of read length in one run is of paramount importance to maximize the amount of sequence information generated per unit time. However, extension of read length in a single non-gradient separation run necessarily means a longer run time. Because separation conditions are maintained constant during the run, a compromise between the quality of the separation and the run time must be made. Excessive resolution of the smaller fragments that differ significantly in size (i.e. the difference between fragments of 100 and 101 bases is 1%) must be "built in" to the separation in order to achieve adequate resolution of the larger fragments that do not differ greatly in size (i.e., fragments 999 and 1000 differ by 0.1%). This compromise means that the separation of both the set of the smallest and the set of the largest fragments in a single nm must take longer than if the set of small fragments was separated under one set of conditions in one column and the set of largest fragments was separated concurrently under a different set of conditions in another column.

With regard to sieving buffers, Zhu et al (U.S. 5,089,111) described several types of polymer solutions useful for size based separations of a variety of biomolecules. However, they did not describe the separation of DNA sequencing fragments. Chin (U.S. 5,110,424; U.S. 5,096,554) describes fractionation of double-stranded nucleic acids by counter-migration capillary electrophoresis through a polymer solution. Grossman (U.S. 5,126,021) described low viscosity polymer solutions for capillary electrophoresis. Dubrow (U.S. 5,164,055) described the use of high viscosity polymer matrix for analysis of proteins and nucleic acids by capillary electrophoresis. Demorest, Werner, and Wiktorowicz (U.S. 5,264,101) described the use of a capillary tube with a charged inner surface for use in capillary electrophoresis. A solution of charged polymer is used in their buffer formulations. Finally, Grossman (U.S. 5374,527) described a specific formulation of a solution of polyacrylamide "sieving buffer" for use in a high-resolution DNA sequencing method. With regard to other sieving matrices used to achieve size-based separations in capillary electrophoresis, Bente and Myerson (U.S. 4,810,456)

described a method of preventing shrinkage defects in electrophoretic gel columns. Karger and Cohen (U.S. 4,865,706, U.S. 4,865,707, U.S. 4,997,537) described methods for preparing high performance micro capillary gels for capillary electrophoresis. Novotny, Dolnik, and Cobb (U.S.5,080,771) described a method of preparing capillary gels formed by spatially progressive polymerization using migrating initiator. Shieh (U.S. 5,098,539) described a method of preparing a gel-containing micro capillary column. Holloway (U.S. 5,110,439) described a capillary gel column with a coupling layer. Schomburg, Lux, and Yin (U.S. 5,141,612) described improved methods of preparing polyacrylamide gel-filled capillaries for separation of DNA fragments. Guttman (U.S. 5,213,669) described a capillary column containing a dynamically cross- linked gel composition for separation of proteins by capillary electrophoresis. Guttman (U.S. 5,332,481) described a method of filling an internally coated capillary with a gel in its polymerized state without damaging the gel. Konrad and Pentoney (U.S. 5,273,638) described a method of DNA sequencing using the products of ddNTP/polymerase/primer reactions and capillary electrophoresis. They described the use of a single fluor-labeled primer and varying relative concentrations of 3 ddNTP's to encode the various dideoxy-terminated fragments by the relative intensities of the peaks; the presence of the fourth base was inferred by the absence of a peak in the separation due to the absence of the corresponding ddNTP in the reaction mixture.

Extension of the read length in gel electrophoretic separation of sequencing fragments has concentrated primarily on increasing the separation length of the electrophoretic gel. For example, Stegemann et al. (Methods MoL Cell. Biol. 19923(1) 53-55) extended resolution by 150 bases by increasing the gel separation distance from 190 to 250 mm. Stegemann et al. (Methods MoL Cell. Biol., 19912(4) 182-184) also found that by varying separation distance on ultra-thin gels, they could extend the read length of 350-500 bases on a 30 cm gel to 600-800 bases on a 50-cm separation gel. Zimmerman et al (BioTechniques 17(2) 1994 302-306) improved read length to 1000 bases by a

combination of optimization of nucleotide ratios and use of high concentrations of detergents in the sequencing reaction mixture in combination with use of thermostable enzymes in a cycle sequencing protocol. Ansorge et al. (J. Biochem. Biophys. Methods 10(3-4) 1984 237-243) described another common strategy for increasing the number of the number of resolvable bases per gel by employing an electrical field gradient along the length of the electrophoretic gel*

SUMMARY OF THE INVENTION This invention encompasses a novel method for nucleic acid sequencing that results in a significant increase in efficiency over prior art approaches. The preferred strategy focuses on substantially increasing read length and sequencing throughput by combining controlled sequencing reaction conditions with an array of short electrophoretic separations to allow very rapid separation and detection of nucleic acid sequencing products over a large number of bases of contiguous sequence.

In particular, the invention encompasses a method for sequencing nucleic acids comprising:

(a) conducting multiple terminator base/primer extension reactions which terminate on different regions of a nucleic acid to be sequenced wherein reactions corresponding to each region are in different containers and wherein the terminator base or primer are labeled to generate a series of labeled terminated nucleic acid fragments corresponding to each primer extension reaction;

(b) simultaneously or independently separating the labeled terminated nucleic acid fragments from each region; and

(c) analyzing the labeled terminated nucleic acid fragments from each region to determine the nucleic acid sequence of each region thereby determining the nucleic acid sequence of the nucleic acid to be sequenced.

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Alternatively, an extension reaction throughout the length of the nucleic acid to be sequenced may be run and then the labeled terminated sequences from specific regions are separated by sieving polymers or buffers or on acrylamide gel. These labeled fragments from the different regions are then separated.

Thus, either the primer or the terminating base (or both) may be labeled and the separation is preferably conducted by simultaneously transferring to and separating the labeled terminated nucleic acid fragments on capillary electrophoresis columns. Another object of the invention is to provide a method for using a series of short capillaries containing electrophoresis sieving media to optimize separation of nucleic acid sequencing fragments of well-defined, overlapping size ranges, each size range typically spanning at least 100 bases, and the combined series nominally spanning up to and exceeding 1,200 bases. Yet another object of the invention is to provide methods for establishing compatible dideoxy-terminated nucleic acid sequencing reaction conditions capable of generating multiple overlapping distributions of sequencing products, each distribution range typically spanning at least 100 bases, and the combined series of distributions nominally encompassing up to and exceeding 1,200 bases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates the principle underlying the sequencing/separation methodology, whereby extended read length on a template DNA is achieved by dividing the sequencing reaction and separation steps into a series of overlapping subsets of fragments, each subset being rapidly separated in an electrophoretic separation optimized for that subset of fragments.

Figure 2A-C shows a schematic of how the various components of the invention interact.

Figure 3A shows a cross-sectional view of several sample handling plate wells.

Figure 3B shows a schematic of liquid flow from sipper capillary to sample handling plate well. Figure 4 shows flow of sample into sipper capillary.

Figure 5 shows sample handling plate with sample handling plate well and waste and primary buffer wells.

Figure 6 shows top plan view of a preferred separation plate.

Figure 7 shows a cross-sectional view of the preferred separation plate.

Figure 8 shows a schematic diagram of the optical system for reading capillaries.

Figure 9 shows the separation of two distributions of single-stranded DNA fragments by capillary electrophoresis. Figure 10 illustrates individual optimized separations of two distributions of single-stranded DNA fragments such that separation of both occurs in a comparable time period.

Figure 11A and 11B show the separation of two subsets of DNA sequencing fragments produced from common template DNA such that in one sample, sequencing fragments extend essentially over one distribution of sizes (lengths) and in the second set, the sequencing fragments extend over essentially a different distribution of sizes; however, there is a common region of overlap of sizes between the two distributions.

Figure 12A and 12B show the region of overlap of sizes in the two distributions and shows alignment of the common fragments.

DETAILED DESCRIPTION OF THE INVENTION

The primary performance specifications for a sequencing method are throughput (bases per hour) and read length (bases). Higher throughput increases the productivity of the sequencing step. Longer read length reduces the number of required templates, thereby reducing the preparation and

processing time which precedes sequencing and the post-processing required to assemble fragment sequences. Previous attempts to achieve long read lengths have generally relied upon increasing the length of the separation medium with a resulting increase in separation times, severely compromising throughput Efforts to achieve faster migration on shorter separation media through increased field strengths are limited by the degradation of resolution caused by heating. In addition, the use of longer separation media means that one has to develop separation conditions driven by the resolution of the largest fragments (1 base out of 1000 requires 0.1% resolution). Maintaining such high resolution over a large range of fragment sizes is difficult and results in a degree of resolution of smaller fragments that is unnecessary. The approach of the instant invention avoids these problems by using a conceptually simple, yet unexpectedly powerful, strategy. To illustrate the advantages of this invention, it is useful to refer to the gradient sequencing gel as a device for balancing speed of migration with the range of molecules resolved. The approach in this invention is a step-gradient gel in which the different regions of the gradient have been separated into individual short gels. When sample is loaded onto each of the individual gels and run, the same overall range of molecules is resolved as on a single gradient gel, but running the set of gels in parallel greatly increases speed of analysis and therefore the throughput

Figure 1 illustrates the basic principles underlying the sequencing and separation methodology of the invention. For purposes of illustration, a fragment of template DNA 65 bases in length is used, as shown in the sequence data across the top of Figure 1. The four individual nucleoside bases are coded as a set of four types of lines representing electrophoretic bands, as illustrated beneath the DNA sequence information at the top of Figure 1. Rather than attempting to sequence the entire length of this DNA in a single run, which would require a extended length of time as illustrated by the horizontal arrow in the figure, the sequencing task is divided into 3 subsets of slightly overlapping fragments as shown in the bottom half of Figure 1. These 3 subsets of fragments are produced by proper manipulation of the Sanger dideoxy-mediated

sequencing reactions such that fragments spanning 3 discrete regions of the original template DNA are produced. These subsets of fragments are then simultaneously separated in rapid parallel electrophoretic separations, with each separation optimized for resolution of the relatively narrow distribution of fragments contained in each subset of fragments. The subsets of sequence information obtained from these separations are then used to reconstruct the sequence of the original template DNA based on the overlap of the sequence data in the 3 subsets of fragments.

Methods and apparatus for conducting nucleic acid sequencing separations described in the application are described in an application entitled "Capillary Electrophoresis Apparatus and Method", U.S. Serial No. 08/408,683, filed March 21, 1995, and assigned to the same Assignee as this application. That application, in its entirety, is incorporated herein by reference. Device and methods for sample acquisition, processing, injection, separation and detection are discussed briefly below; details of the various components can be found in the aforementioned application.

Figures 2A-C show the interaction of various parts of the electrophoresis system of the invention. Figure 2A shows the sample handling plate 21 with an array of sample handling wells 74 with an corresponding array of sipper capillaries 22,. The array of sipper capillaries is aligned with wells of a multiwell plate j& which contain samples 31- When the sipper capillaries SZ are in the sample, an aliquot of sample is transferred to the sipper capillary by wicking action. The sample handling plate 21 is then moved to base plate 22. as shown in Figure 2B. Sample handling plate 21 and base plate 22 fit together to form a sealed inner chamber £9. which can be pressurized or evacuated through port 22- In this way, the samples in capillaries jJ can be manipulated and eventually presented in sample handling wells 24 for electrophoresis. Figure 2C shows how the electrophoresis separation plate(s) 100 containing an array of electrophoresis capillaries 10 . are aligned with the sample handling plate wells 4.

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Figure 3 A shows a cross-sectional view through several wells 74,. The sample handling plate 21 is assembled from a sampling block 21 which defines the funnel shaped base wells with openings 22- Mixer block 28 has passages 22 which are aligned with openings 22- The mixer block 2S and sampling block 25 are separated by a porous matrix such as membrane SQ. Aligned with mixer block 25 is sipper block 21 with sipper capillaries $2. 82a is filled with sample and 52b is not The sipper block 2 mixer-block 78. and sampling block 21 are fastened together so that a channel a-b is defined which is interrupted by the membrane jJQ as shown in Figure 3B. Membrane j is typically made of a wide variety of porous matrix materials where, for most applications the porous matrix materials should have little or no affinity for sample. Useful porous matrix materials include membrane materials such as regenerated cellulose, cellulose acetate, polysulfone, polyvinylidine fluoride, polycarbonate and the like. For DNA samples, a cellulose acetate membrane such as that available from Amicon is useful.

Figure 4 illustrates the flow of sample S from a well of a multiwell plate into sipper capillary j£. Thus the ends of the array of sipping capillaries 22 on sample handling plate 21 are dipped into samples contained in an array of samples such as a 96 well plate and the samples are metered into the sipping capillaries by capillary action. At this point, the sample handling plate 21 with its sipper capillaries filled with samples is placed on base 22 to form a sealed inner chamber £2, Figure 2B.

Figure 5 illustrates the sample well 24 flanked with a well for waste electrophoresis buffer 3Q from a previous separation and a well for fresh run buffer 31 which is deposited from a capillary 1Q1 during a flushing before injecting presented sample from the sample handling well into the capillary 105 and then used during the electrophoresis separation. The capillary Q addresses the waste 20, buffer 51 and sample 2A positions by moving the separation plate with respect to the sample handling plate for electrophoresis. Figure 6 shows the electrophoresis separation plate ifiQ having 8 capillaries 1Q1 mounted on a frame _1£2; upper buffer reservoir Q2. provides

buffer to the capillaries 101. Orientation notch 107 provides a means for aligning the separation plate for transferring sample or reading columns. Electrode UM and an electrode at the injection end of each of the separation capillary iflfi provide for electrical communication through the buffer. Figure 7 is a cross-sectional view through a separation plate.

Thus, in operation, samples from an array of samples 21 such as a multiwell plate are wicked into an array of sipping capillaries 22 of the sample handling plate 21. The sample handling plate 21 is placed on base 22 and the sample is manipulated by pressurizing the chamber ££ defined by the sample handling and base plates and finally moved to the base plate wells 24 for presentation to the capillaries in the separation plate 100. However, prior to transferring the sample to the capillary, the capillaries are washed with buffer and primed with buffer. Samples are injected into the capillaries and electrophoresis is conducted in the capillaries in the separation plate. When the electrophoresis is finished, the separation plate may be moved to an analysis station. The over all scheme is shown in Figures 2A-C. After electrophoresis, the separation plate can be stored or read as shown in Figure 8.

Capillary electrophoresis columns can be analyzed in a variety of ways including the methods shown in U.S. Patents 4,675,300, 4,274,240 and 5324,401. The sample injection and separation are conducted in one location and the plate may be transported to a different location for analysis. Figure 8 shows a block diagram of one optical system for reading the capillaries. Power supply 3J} energizes the photomultiplier tube 21- Power supply 22 energizes a 75 watt Xenon lamp 21- Light from the lamp 22 is condensed by focusing lens 3 which passes light to the excitation filter 21- A dichroic mirror 26. directs excitation light to microscope objective 21- The separation plate IQQ with capillaries 101 is mounted on a rectilinear scanner to pass the capillaries over the light from the microscope objective 22- Adjustable slit 12 defines the image of the inner bore of a capillary on the active element of photomultiplier tube

21. Signal from photomultiplier tube 21 is fed to analog-to-digital convener 53. the output of which is stored in computer &.

Those skilled in art will recognize a number of different types of separation media suitable for practicing this invention. For example, a set of short electrophoresis slab gels can be employed as the separation media. In this case, each slab gel in the set would be tailored for optimum resolution of a rather narrow range of sizes of DNA sequencing fragments, specifically the range corresponding to the range of sizes produced in any one of the set of DNA sequencing reactions described below. Instead of conventional slab gels, it is desirable to use a set of short electrophoretic columns (such as illustrated in Figure 6), each containing a replaceable sieving buffer optimized for a particular size range and the sequencing products generated from the template DNA will be resolved in parallel on the set of columns. Alternatively, a set of capillary electrophoresis capillaries filled with acrylamide gel can also be used in place of the capillaries with replaceable sieving buffers. Either of these will enable rapid production of a set of overlapping sequences that together yield a long read length. That each of the individual sieving buffers is homogenous (as compared to a gradient) simplifies replacement of the sieving matrix. Automated sample loading results in short processing times and coupled to the high degree of parallelism achieved by arranging columns in a 96 well format enables generation of long read-length sequence data on multiple (8 or 12) template DNA samples. In this way, long read lengths can be obtained rapidly, reagent consumption is minimized because capillary electrophoresis separations require minute (nanoliter) sample volumes, and separation conditions are optimized for each of the defined and relatively small read windows. Likewise, one set of capillary electrophoresis capillaries such as illustrated in Figure 6, each filled with a different sieving medium (acrylamide gel or sieving buffer) such that each is capable of optimally separating one of the subset of sequencing fragments of various size distributions described above could serve as the separation medium.

Alternatively, a single acrylamide slab gel prepared in such a way that a lateral sieving gradient is formed across the width of the gel such that lane

1 of the gel is optimized for the subset of smallest sequencing fragments, lane

2 optimized for the next smallest subset of fragments, etc., with the last lane optimized for the largest subset of sequencing fragments of various size distributions described below could be employed as the separation medium. In this way, conventional DNA sequencing instruments could benefit from some of the advantages of this invention.

Alternatively, a set of electrophoretic slab gels could be tailored such that each gel in the set was formulated for optimum resolution of a rather narrow range of sizes of DNA sequencing fragments, specifically the range corresponding to the range of sizes produced in any one of the set of DNA sequencing reactions described below. Each of the set of slab gels could then be run on a separate DNA sequencing instrument such as the Applied Biosystems Model 373 DNA Analysis System or the Pharmacia A.L.F. DNA Sequencer. Data from the series of gels could then be combined to reconstruct the sequence of the template DNA while achieving the extended read lengths obtainable with this invention.

Any of a number of capillaries can be used for the separation process. Fused silica capillaries - 100 mm in length and with inner diameters of -25 to 100 microns are preferred. Smaller id capillaries produce less Joule heating during the separation process compared to larger id capillaries and thus can be run a higher electrical field strengths relative to their large bore counterparts. Higher operating field strengths reduce the overall run time. However, loading of the somewhat viscous solutions of sieving polymers into the small bore capillaries is more difficult than loading such solutions into large bore capillaries. In addition, deteαion of the separation bands in the small bore capillaries is more difficult than for large bore capillaries since a proportionally smaller quantity of sample must be loaded into the small bore capillary to avoid overloading the separation matrix which would result in a loss

of resolution between adjacent bands. Thus, the choice of the proper id of the capillary will be a compromise between the aforementioned factors.

The inner wall of the silica capillary may be derivatized via some chemical agent to suppress electroosmotic flow in the capillary during the separation as well as passivate the inner wall of the silica to prevent irreversible adsorption of sample species onto the capillary wall; this latter factor results in asymmetrical (tailed) bands for the sample species as well as loss of sample components onto the wall of the capillary with a concomitant loss of detection sensitivity. The preferred chemical agent for derivatizing the inner wall of the separation capillaries is a covalently attached layer of linear polyacrylamide attached to the silica via an intermediate silane coupling agent Such chemistry is well known in the literature (e.g., see Hjerten, U.S.4,680,201; Nσvotπy, U.S. 5,074,982). Hjerten describes treatment of the capillary with methacryloxypropyl-trimethoxysilane followed by in situ polymerization of the linear polyacrylamide attached to the silica wall via the intermediate silane. Novotny describes use of sulfonyl chloride the convert SiOH to SiCl. followed by treatment of this surface with a vinyl Grignard reagent to form a coating of monomer on the surface of the silica. This is followed by in situ polymerization of the linear polyacrylamide attached to the silica wall via the intermediate monomer functionality . Either of the methods is adequate for derivatizing the silica wall; other silane and silicone agents as the primary coupling agent are also adequate. In addition, other linear polymers such as polyvinylpyrollidone as the coating attached to the silica wall are also acceptable substitutes for the linear polyacrylamide. The preferred background electrolyte in the electrophoretic separation media is IX TBE (89.5 mM Tris, 89 mM borate, 2 mM EDTA, pH -82); this supporting electrolyte is used extensively in electrophoresis gels used for DNA sequencing (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, NY, 1989. Chapter 13.) as well as for sequencing in capillary electrophoresis media. Other electrolytes useful for the separation media are TAE, TSE, etc. which substitute

acetate or sulfate for the borate in the electrolyte. In addition, the preferred separation medium will contain 7-8 M urea as a denaturing agent to maintain the DNA fragments in a single-stranded form during the separation process as well as suppress hairpin loops due to self-complementarity within individual fragments. pH of the separation media can vary from pH 9 to pH 7.5, with the preferred pH range of 8 to 8 .

A novel feature of this invention is the use of a series of sieving matrix formulations that will yield the requisite resolution of the various distributions of sequencing fragments in a comparable time period. For example, it is desirable to achieve the resolution of 900 -1020 base fragments in a sieving medium in one separation in a time period comparable to the separation of 10-120 base fragments in another sieving medium in a concurrent separation.

The separation matrix is based on sieving of the various fragments to achieve a size-based separation. To form the sieving matrix, a porous polymer network must be formed as the separation matrix. The preferred polymer for this sieving matrix is polyacrylamide as there is substantial prior art indicating the utility of this polymer for sequencing separations. However, other polymers, such as polyvinylpyrollidone may also be useful for achieving the desired separations.

Several properties of the polymer may be adjusted to achieve the resolution of the various distributions of sequencing fragments in the desired common time period. The molecular weight of the strands of linear polymer can be adjusted to produce sieving media with different pore sizes which would be useful for separating the various distributions of sequencing fragments of different sizes. In addition, the overall concentration of the linear polymer in the sieving solutions can be adjusted to vary the size of the transient pores in the sieving matrices. Finally, the polydispersity of the linear polymer is responsible for the distribution of pores sizes in a given sieving solution of linear polymer of fixed concentration and nominal molecular weight It is anticipated that adjustment of this parameter will allow optimization of the

dynamic range of separation of a specific formulation, e.g. resolution of 100 or 120 or 150 fragments in a given time period.

Other additives incorporated into the separation medium might also prove useful for optimizing the separations of the sequencing fragments. For example, formamide might be added to reduce the viscosity of a given sieving matrix, making it easier to load into the small diameter capillaries. In addition, use of formamide could improve resolution of the various fragments by eliminating GC compressions which are common in sequencing separations. Those skilled in the art of electrophoretic separations will recognize that a broad range of electrical field strengths can be used to accomplish the separations. High separation field strength is desirable since migration velocity of the sequencing fragments through the sieving matrix is essentially proportional the field strength and thus separation time is reduced. However, Joule heating in the sieving matrix, which is related to the product of voltage and current in the sieving matrix, increases dramatically with field strength and, when excessive, can degrade or destroy the separation due to thermally induced convective mixing in the sieving matrix. Another factor which gives rise to degradation of the separation as field strength is increased is the onset of reptation of the sequencing fragments through the porous separation matrix. When the separation occurs under conditions where reptation is operative, resolution of adjacent fragments is severely deteriorated since the fragments are not migrating through the sieving matrix on the basis of size. Generally, field strengths up to 500 V/cm might be useful to accomplish these separations; however, field strengths between 200 to 300 V/cm are preferred.

Description of Sequencing Procedure

The initial step in the sequencing process is to produce template nucleic acid whose sequence is to be determined. This can be done by a variety of methods including cloning, PCR™ amplification, etc.; methods suitable for production of this template nucleic acid are described by Sambrook et al (Sambrook, J., E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory

Manual, Cold Spring Harbor Press, NY, 1989). The template nucleic acid may be either single- or double-stranded; the preferred form is single-stranded nucleic acid. Methods of converting double-stranded nucleic acid to the single- stranded form are described by Sambrook et al The single-stranded template nucleic acid is annealed with a primer, typically by warming the primer/template mixture to 65-75 °C and then slowly cooling the warmed mixture to -30 °C to allow formation of a stable hybridization product. The annealed mixture is then chilled on ice to inhibit dissociation of the hydribization product. Next the dideoxy-mediated sequencing reactions are run on the annealed primer/template mixtures. Typically, a set of 4 dNTP's and appropriate ddNTP's (depending on the type of terminator used) are added to the annealed template/primer along with a DNA polymerase enzyme; suitable dNTP's, ddNTP's, and polymerase enzymes are discussed below. The reactions are conducted at 37 °C for a period of time varying from several minutes to perhaps 30 minutes, depending upon the reaction conditions. The sequencing reaction mixtures are halted at the proper time by addition of a stop solution, typically 95% foπnamide containing millimolar concentrations of EDTA. The DNA sequencing fragments are next denatured from the duplex form by heating the sequencing reaction products to 75-95 °C for several minutes and then chilling the denatured mixture quickly on ice to 0 °C to inhibit renaturation. At this point, the sequencing fragments are ready for loading onto the electrophoretic separation medium and subsequent size-based separation using the electrophoretic separation means described above. The method of the instant invention differs from the prior art in that instead of generating a full set of sequencing fragments spanning the full length of the template DNA in a single reaction (or set of 4 complementary reactions, one for each of the nucleoside bases) to accomplish an extended read length in a single separation, the full set of sequencing fragments is broken up into slightly overlapping subsets of the full set of sequencing fragments by running multiple, controlled sequencing reaαions on smaller amounts of the template

DNA. For example, rather than running a single sequencing reaction on 12 pmole of template DNA to produce sequencing fragments from 10 to 1200 bases in length, the method of this invention could run 12 individual sequencing reactions, each on 0.1 pmole of template DNA to produce the same quantities of the sequencing fragments. Because the separation method of the invention uses optimized separation media for rapid separations of each of the samples, the bands in the separations will remain sharp, thus improving detectability of ti e individual fragments; thus the method of the instant invention may not require 12 x 0.1 pmoles of template but may be implemented with smaller quantities (e.g., 12 x 05 pmoles) of template DNA. These subsets of fragments are then rapidly separated in parallel on an optimized set of electrophoretic media such that the full sequence of the template DNA can be reconstructed from the various subsets of sequence information generated above. Various aspects for implementing the sequencing reaction and separation steps are discussed below.

Those skilled in the art will recognize a large number of methods of controlling sequencing reactions. Several faαors are important in determining the rate and extent of polymerization of the polymerase enzyme during the sequencing reaction. The reaction temperature at which the polymerization extension reaction is carried out determines the Michaelis/Menton rate constant of the enzyme. The concentration of cofactors such as Mg* ² or Mn ⁺² ions and the pH of the reaαion buffer govern the reaction rate. Finally, the concentration of monomers (dNTP's and ddNTP's) used in the synthesis of the DNA sequencing fragments can control the rate of the reaction. Each of these faαors may be used to produce the desired set of sequencing fragments of slightly overlapping size distribution.

Control of the reaαion kinetics to produce the desired stagger and overlap of the sequencing fragments can be accomplished in a number of ways. For example, for a given template DNA, the extension/termination reaαions could be initiated in a set of 8 or 12 reaαion mixes containing the template and the full complement of requisite dNTP's needed to produce fragments over the

entire size range of template in each mix; production of the set of partially overlapping distributions of fragments of different size distributions could be accomplished by sequential addition, at appropriate intervals during the course of the reactions, of appropriate concentrations of the various ddNTP's to the series of reaction mixtures such that each mixture is terminated at a different stage during the extension reaαion. Alternatively, each of the set of reaction mixtures can initially be run under reaction conditions with limiting concentrations of the 4 dNTP's; each reaαion mix would then be extended until exhaustion of the dNTP's to yield non-terminated fragments of the same size distribution. At that point another limiting dose of dNTP's would be added to all the reaction mixtures and an appropriate concentration of the ddNT s added to only one of the reaαion mixtures. Thus, chain extension would restart in all of the mixes, and the one mix with the ddNTP would also begin the termination reaction to produce the smallest size-distribution of fragments. This process would be cyclically repeated on the remaining set of reaction mixtures; during each cycle, one of the mixes would receive the dose of ddNTP terminators to produce the next largest distribution of terminated fragments. Alternatively, all reaction mixtures in a set of 8 or 12 reaction mixes can be initiated with the full complement of requisite dNTP's needed to produce fragments over the entire size range of template in each mix; production of the set of partially overlapping distributions of sequencing fragments of different size distributions could be accomplished by addition of appropriate chemical agent to pause the extension reaαion in each well after a seleαed interval of extension; EDTA is such a chemical agent and it would chelate the Mg* ³ or Mn ⁺² cofattor essential for the DNA polymerase to function. After each of the reaction mixtures has been extended to the desired length and polymerization has been paused by addition of this agent an appropriate dose of the ddNTP's is added to each of the reaαion mixtures. The polymerase enzyme is then activated again by addition of an amount of Mg* ² or Mn ⁺² cofaαor in molar excess relative to the EDTA, such that unchelated divalent metal ion is

available as cofaαor for the polymerase enzyme to reinitiate the extension/termination reaαion.

Alternatively, temperature could be used to pause the reactions in the preceding protocol. Each of the mixtures described above would be quick- chilled at a seleαed interval after initiation of the extension reaction to pause the polymerase enzyme in that particular mύcture. After all reactions have been paused at the proper intervals, ddNTP terminators are added to all reaαion mixtures and the extension/teπnination reaαion is initiated by warming the mixtures to reactivate the enzyme.

Extension/Termination Reaction Components

Those skilled in the art recognize numerous ways in which detection labels can be incorporated into the DNA sequencing fragments. For example, the primer used to initiate the DNA polymerase extension reaction can be labeled on the 5' end with an appropriate fluorophore such as fluorescein, JOE, FAM, ROX, Texas Red, FTTC, NBD, etc. (see Smith et al., Nature 1986321(12) 674-679). Alternatively, the fluorescent label could be on the 5' terminus of the primer could be an infrared-fluorescent-label such as IR-144 laser dye (Middendorf et al , J. Cell Biol. 1991 115 81N). A fluorescently-labeled dNTP such as Fluore-dATP (fluorescein-dATP, Pharmacia, Inc., Piscataway, NJ), could be used to incorporate label throughout the sequencing fragment chains during the extension/termination reaction. Fluorescently-labeled chain extension terminators such as those described by Prober et al. (Science 1987238 336-341) could also be used to produce the desired label. An alternative and less preferred method of labeling the sequencing fragments would be incorporation of radiolabels such as ³² P or ³ⁱ S in the sequencing fragment .chains via use of the appropriate radiolabeled dNTP (e.g. α- ³² P-dATP).

Numerous enzymes such as the Klenow fragment of E. coli DNA Polymerase I, AMV (Avian Myeloblastosis Virus) Reverse Transcriptase, Taq (Thermus aquaticus) DNA Polymerase, Bca DNA polymerase, Tth (Thermus thermophtlus) DNA Polymerase, or modified analogs of T7 DNA Polymerase

such as Sequenase ™ Ver 1.0 or Ver 2.0 may be employed to accomplish the chain extension/ termination reaction; a preferred enzyme would be one of the modified T7 DNA polymerases such a Sequenase™ Ver. 2.0, since they have a high tolerance for accepting modified nucleoside triphosphates in the chain extension/termination reaction and, when used in conjunction with Mn ⁺² ion as cofaαor in the reaction matrix, they produce a more uniform distribution of fragments for the longer fragment sizes.

Any of a number of possible primers are amenable to use in the present invention. Charaαeristics of the primer necessary for implementing the methods of the instant invention are: 1) free 3'-hydroxyl group on the primer to allow chain extension by the polymerase, 2) complementarity to the 3' end of the template DNA, 3) sufficient length to form a stable hybridization produα with the template DNA, and 4) attached labels, if any, must not interfere with the hybridization reaction or the chain extension/termination reaction. Possible primer lengths can range from about 6 nucleotides (hexamer) to 20-30 bases; the sequence of the primer is most likely determined by a complementary sequence in the cloning veαor used to produce the template DNA. Typical sequences can be derived from cloning veαors such as any member of the M13 family (M13mpl8, M13mpl9, etc.), the pUC family (pUC18, pUC19), the pTZ family, etc. A preferred primer is based on the M13 primer (-40) sequence 5'-GTTTTCCCAGTCACGAC-3'. Components for the chain extension/termination reaction include the four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) or appropriate analogs (e.g. 2'- deoxyinosine-5-triphosphate (dlTP), 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP), Pharmacia's Fluore-dATP) and dideoxyribonucleoside triphosphates (ddATP, ddCTP, ddGTP, ddTTP) or analogs (e.g., cordycepin, DuPont dye-labeled terminators T-526, C-519, A-512, G-505, etc.)

The coding of the dideoxy-terminated fragments from the A, C, G, and T reactions can be accomplished in a variety of ways. A preferred method is to utilized four distinct fluorescent dyes, each corresponding to one and only one of the four nucleoside bases in DNA. The four dyes possess different

fluorescent emission maxima, preferably with minimal overlap of emission wavelength. The fluorophores could be incorporated into the sequencing fragments either by use of 5'-labeled primers or via use of fluorescently-labeled dideoxy-based chain terminators. Alternatively, tile use of two fluorophores in either the labeled primer or labeled terminator method could be employed; in this method, two fluorophores can be used to distinguish among 4 nucleoside bases by varying the relative amounts of label incorporated into the fragments such that for instance, the T-terminated fragments are 2X or 3X more intense than the A-terminated fragments even though the same fluorophore is used to label both. The same strategy would be applied to the C-teπninated and the G-terminated fragments, although a second fluorophore dye would be used to produce those fragments. Thus, the set of four terminated fragments are distinguishable by a combination of color and relative intensity of their bands (see H. Swerdlow et al, Analytical Chemistry 1991 63 2835-2641; W. Ansorge, DE 3841 565A1; Konrad and Pentoney, U.S. 5,273,638).

EXAMPLES

EXAMPLE 1 Separation of a Set of Fluoresceπtly-Labe d Single-Stranded DNA Fragments

With Unit Base Resolution in a Replaceable Sieving Matrix

The separation of a series of 5'-FAM-poly dA, fragments, with X ranging from pdA, ₀ to pdA^ and where each fragment in the series differs by a single base, is demonstrated on a 10-cm long capillary with an effective separation length of 7 cm (point of injection to point of detection). The fragments were produced by enzymatic extension of a pdA ₁₀ oligonucleotide which bad been chemically labeled on the 5'-end with FAM (fluorescein) fluorophore. The extension reaαion was done in a 50 μL volume which contained 200 mM potassium cacodylate, pH 7.2, 4 mM MgCl ₂ , 1 mM β- mercaptoethanol and 50 mM 5'-FAM-pdA, ₀ (Keystone Laboratories, Menlo

Park, CA). To this reaction mixture were added 2.63 μL (50 units) of terminal transferase enzyme (Sigma Chemical Co., St. Louis, MO) and 1 μL of 50 mM dATP (Sigma Chemical Co., St. Louis, MO). The mixture was incubated for 1 hour at 37 °C, after which 12.5 μL of the reaction m ture was removed and the removed aliquot chilled on ice in the dark to inhibit further reaction. Next 1 μL of 50 mM dATP, 2.5 μL of 1 M potassium cacodylate, 5.11 μL of distilled, deionized water, 1.25 μL of 40 mM MgC12, 0.1 μL of 100 mM β mercaptoethanol, and 2.63 μL (50 units) of terminal transferase was added to the remaining original reaction mixture. This was again incubated for 1 hour at 37 °C, after which a 12.5 μL aliquot of the reaction mixture was removed and chilled on ice in the dark to inhibit further reaction. Next, 1 μL of 50 mM dATP, 333 μL of 1 M potassium cacodylate, 7.9 μL of distilled, deionized water, 1.67 μL of 40 mM MgCl ₂ , 0.2 mL of 100 mM 3-mercaptoethanoL, and 2.63 μL (50 units) of terminal transferase was added to the remaining original reaction mixture. This was again incubated for 1 hour at 37 °C, after which a 25 μL aliquot of the reaction mixture was removed and chilled on ice in the dark to inhibit further reaαion. To the remaining original reaction mixture were added 1 μL of 50 mM dATP, 5 μL of 1 M potassium cacodylate, 13.6 μL of distilled, deionized water, 2.5 μL of 40 mM MgCl ₂ , 025 μL of 100 mM β- mercaptoethanol, and 2.63 μL (50 units) of terminal transferase. Incubation of this mixture was carried out for 1 hour at 37 °C, after which the mixture was chilled on ice in the dark. Portions of the 4 aliquots produced above were blended together in water to yield the composite sample used below.

The combined sets of fragments generated above were then analyzed on a 10 cm length of 100 micron id fused silica capillary which had been covalently coated with a layer of linear polyacrylamide via the procedure described by Hjerten (U.S Patent 4,680,201). The capillary was filled with a solution of sieving buffer which consisted of 13% w/v linear polyacrylamide in a IX TBE buffer (89.5 mM Tris base, 89.5 mM boric acid, 2 mM EDTA, Na ₂ \ pH 8.3). The 13% linear polyacrylamide solution was prepared by dissolving 0.65 g of acrylamide (Sigma Chemical Co., St. Louis, MO) in 5 mL of IX TBE

buffer. This solution was then degassed for 45 minutes under a vacuum of 25 inches water. The polymerization reaction was then initiated and catalyzed by addition of 10 μL of 10% (w/v) ammonium persulfate (Sigma Chemical Co., St. Louis MO) and 5 μL of TEMED (N,N,N',N'-tetraethylmethylenediamine, Sigma Chemical Co., St. Louis, MO). After brief mixing, the reaction mixture was loaded into 0.5 mL hypodermic syringes (Beαon Dickinson and Co., Franklin Lakes, NJ), capped, and stored in a refrigerator overnight to ensure complete polymerization.

The capillary was attached to the polyacrylamide-filled syringe via a 2 cm section of Teflon™ tubing (Cole Parmer Instrument Co., Chicago IL) and the sieving matrix loaded into the capillary from the syringe. The ends of the capillary were then immersed in reservoirs of IX TBE running buffer in a separation plate similar to that in Figure 6. Detection was accomplished as described in Figure 8 with a stationary detection point 15 cm from the injection (cathode) end of the capillary. A 2 μL aliquot of the combined 5'-FAM pdA, DNA fragments was loaded into the injeαion well of the separation plate and electroinjection was run for 20 sec at 3.5 kV. The residual sample was removed from the injection well via flushing the well with - 1 mL of IX TBE run buffer. Electrophoresis was then conduαed for up to 35 minutes at 2 kV applied potential. Data collection was at 20 Hz on a Perkin Elmer/Nelson Model 1020 data system (PE/Nelson, Cupertino, CA).

The separation and analysis of the combined distributions of 5'-FAM- labeled poly dA, fragments is shown in Figure 9. Separation was accomplished at a relatively low voltage of 200 V/cm in about 20 minutes. Resolution exceeding R, = 1.0 of adjacent fragments was achieved for the fragments of x = 2 out to x = -200 bases. Total concentration of DNA fragments in the injection sample for these separations was - 0.1 micromolar, which is comparable to the concentration of sequencing fragments in a typical DNA sequencing reaction (0.5 pmole primer and gel-loading volume of 5 microliters).

EXAMPLE 2 Separation of Two Distributions of Fluorescently-Labeled Single-Stranded DNA Fragments of Differing Size Distributions in a Comparable Time Period in a Replaceable Sieving Matrix

Figure 10 illustrates the basic concept behind the separation strategy of this invention. Two slightly overlapping sets of 5'-FAM-labeled single- stranded polydeoxyadenosine DNA fragments covering two different size ranges were produced using 5'-FAM-pdA, ₀ as a primer for terminal transferase as described in Example 1. The first set of fragments was prepared by reaction of 35 nmoles of dATP with 1 nmole of 5'-FAM-pdA ₁₀ , to produce an average target extension produα size of 35 bases (5'-FAM-pdA _JJ± ). The second set of fragments was prepared by reacting 60 nmoles of dATP with 1 nmole of 5'- FAM-pdA. ₀ , to produce an average target extension produα size of 60 bases (S'-FAM-pdAβo _± ). The two sets of fragments were then separated by capillary electrophoresis similar to that described above except for the use of two different sieving buffer formulations.

Two sieving buffer solutions were prepared which consisted of 11% or 12% (w/v) linear polyacrylamide in a IX TBE buffer (89.5 mM Tris base, 89.5 mM boric acid, 2 mM EDTA, Na , pH 83). The 11% and 12% linear polyacrylamide solutions were prepared by dissolving 0.55 g and 0.60 g of acrylamide (Sigma Chemical Co., St. Louis, MO) in 2 separate 5 mL volumes of IX TBE buffer. These solutions were then degassed for 90 minutes under a vacuum of 25 inches water. The polymerization reaction was then initiated and catalyzed by addition of 10 μL of 10% (w/v) ammonium persulfate (Sigma Chemical Co., St. Louis MO) and 5 μL of TEMED (Sigma Chemical Co _^ St Louis, MO) to each solution. After brief mixing, the reaction mixture were loaded into separate 0.5 mL hypodermic syringes (Beαon Dickinson and Co., Franklin Lakes, NJ), capped, and stored in a refrigerator overnight to ensure complete polymerization.

For the separation, the capillary was attached to the poiyacrylamide- filled syringe via a 2 cm section of Teflon™ tubing (Cole Paπner Instrument Co., Chicago IL) and the sieving matrix loaded into the capillary from the syringe. The ends of the capillary were then immersed in reservoirs of IX TBE running buffer in a separation plate similar to that in Figure 6. Detection was accomplished as described in Figure 8 with a stationary detection point 7.5 cm from the injection (cathode) end of the capillary. A 2 μL aliquot of the 5'-FAM pdA, DNA fragments was loaded into the injection well of the separation plate and electroinjection was nm for 3-5 sec at 1 kV. The residual sample was removed from the injection well via flushing the well with - 1 mL of IX TBE run buffer. Electrophoresis was then conduαed for up to 20 minutes at 2.5 kV applied potential. Data collection was at 20 Hz on a Perkin Elmer/Nelson Model 1020 data system (PE/Nelson, Cupertino, CA).

Both of the pdA, extension mixtures (5'-FAM-pdA _M±ι 5'-FAM- pdA _w± ) were first sequentially run in the capillary filled with the 12% (w/v) polyacrylamide solution for comparison. An overlay of the separations for these two mixtures is shown in Figure 10A; identification of the fragment size is shown along the abscissa of the plot along with the run time. The separation of the 5'-FAM-pdA ₃ j _± mixture produced a series of fragments ranging from pdA, ₇ to pdAjj, with an average migration time of 11.5 minutes for the 5'-FAM- pdA _w fragment Likewise the 5'-FAM-pdA _»± mixture yielded peaks for fragments ranging from pd _ω to pdA*,, with an average migration time of 15 minutes for the S'-FAM-pdAj _o fragments. The partial overlap of the fragments in each distribution is also evident from this figure. The optimized separations of these two distributions of fragments in comparable time periods is shown in Figure 10 B, C. Figure 10B shows an optimized separation of the 5'-FAM-pdA _3J± distribution in a capillary filled with a solution of 12% (w/v) polyacrylamide in IX TBE/7M urea buffer. The average migration time for the distribution is 12 minutes. Figure 10C shows an optimized separation of the 5'-FAM-pdA _Mt distribution in a capillary filled with a solution of 11% (w/v) polyacrylamide in IX TBE/7M urea buffer. The

average migration time of the distribution is 12.4 minutes, comparable to the time required to separate the 5'-FAM-pdA _3S± distribution illustrated in Figure 10B.

EXAMPLE S

Preparation and Separation of a Set of Two

Staggered, Overlapping Sets of FIuorescently-Labeled Single-Stranded DNA

Sequencing Fragments in a Replaceable Sieving Matrix

M13mp 18 single-stranded template DNA ( 1.4 μg, 0.58 picomoles) was annealed with 1 pmole of fluorescently-labeled M13-Fluoro Primer (5TAM- TGT-AAA-ACG-ACG-GCC-AGT-3', Keystone Laboratories, Menlo Park, CA) in 10 μL of annealing buffer which contained 40 mM Tris-HCl, pH 15, 20 mM MgCl ₂ , and 50 mM NaCl). The above mixture in a 0.5 mL Eppendorf tube was incubated for 2 minutes at 65 °C in beaker of heated water; after 2 minutes, the beaker with the annealing mixture were removed from heat and allowed to cool in the dark to 30 °C slowly over a 1 3/4 hr period. After annealing, the above mixture was centrifuged briefly to colleα the reaction mixture on the bottom of the tube and then immediately chilled to 0 °C on ice. To the ice cold mixture were added 1 μL of 100 mM dithiothreitol and 2 μL of Sequenase™ Version 2.0 T7 DNA Polymerase enzyme (13 units/μL, United States Biochemical, Cleveland, Ohio) and the solution mixed by repetitive uptake using a 10 mL Eppendorf pipettor. The mixture was then stored on ice in the dark. Two separate 0.5 mL Eppendorf tubes were loaded with the deoxyπucleoside triphosphates dNTP's and dNTP/ddTTP (dideoxythymidine triphosphate) used in the extension/termination reactions. To Tube A was added 25 μL which contained the 4 dNTFs, each at 1.6 μM (4 picomoles each of dATP, dCTP, dGTP, and dTTP), and 0.16 μM ddTTP terminator (0.4 picomoles) in 50 mM NaCl. To Tube B was added 2.5 μL which contained only the 4 dNTP's, each present in an 18 μM concentration (45 picomoles dATP,

dCTP, dGTP, dTTP) in 50 μM NaCl. These tubes were capped and prewarmed to 37 °C in a water bath.

To Tubes A and B were added 3 μL of the annealed template/primer/enzyme πiixture prepared above ( = 0.134 picomoles template DNA). Both tubes were then incubated at 37 °C for 5 minutes in a water bath. Then 2.5 μL of a solution of each of the dNTP's (4 picomoles each of dATP, dCTP, dGTP, and dTTP) and ddTTP (0.4 picomoles) in 50 mM NaCl was added to Tube B. Incubation continued for another 5 minutes for both tubes at 37 °C. At this point 4 μL of stop solution consisting of 95% formamide containing 20 mM EDTA was added to each tube to end the polymerase extension reaction. Both tubes were then heated to 75 °C for 2 minutes in a water bath to denature the DNA sequencing fragments; both tubes were then quick-chilled in an ice bath and held at 0 °C in the dark until electrophoretic analysis. The purpose of this experiment is to generate two sets of DNA sequencing fragments. One set of fragments (Tube A) covers the range from essentially the primer length (18 bases) to - 100 bases in length. This is accomplished by limiting the molar quantities of the 4 dNTFs in the reaction mix to prevent extension of the primer to very long fragments since the extension reaction will consume all the dNTFs in the reaction mixture while producing fragments up to - 100 bases in length. These fragments will be properly terminated since the ddTTP is present in the reaction mixture from the start of the polymerase reaction. The second set of fragments will be of longer sizes since the initial extension reaction is done in a mixture which does not contain any terminator (ddTTP) but does contain sufficient dNTP's to generate unterminated fragments of sizes comparable to those produced in Tufee A. After the dNTP's are exhausted from this reaction mixture, this set of fragments in then extended and terminated by addition of sufficient dNTFs and terminator ddTTP to produce fragments covering a different but slightly overlapping size range. Again, only enough dNTP's and ddTTP terminator are

added to allow extension for a predetermined amount based on the mole ratios of primer, dNTP, and ddTTP.

The two sets of fragments generated above were then analyzed on a 10 cm length of 100 micron id fused silica capillary which had been covalently coated with a layer of linear polyacrylamide via the procedure described by

Hjerten (U.S Patent 4,680,201). The capillary was filled with a solution of sieving buffer which consisted of 10% (w/v) linear polyacrylamide in a IX TBE buffer (89.5 mM Tris base, 89.5 mM boric acid, 2 mM EDTA, Na,*, pH 83). The 10% linear polyacrylamide solution was prepared by dissolving 0.5 g of acrylamide (Sigma Chemical Co., St. Louis MO) in 5 mL of IX TBE buffer. This solution was then degassed for 45 minutes under a vacuum of 25 inches water. The polymerization reaction was then initiated and catalyzed by addition of 10 μL of 10% (w/v) ammonium persulfate (Sigma Chemical Co., St Louis MO) and 5 μL of TEMED (Sigma Chemical Co., St Louis, MO). After brief mixing, the reaαion mixture was loaded into 0.5 mL hypodermic syringes (Beαon Dickinson and Co., Franklin Lakes, NJ), capped, and stored in a refrigerator overnight to ensure complete polymerization.

The capillary was attached to the polyaαylamide-filled syringe via a 2 cm section of Teflon™ tubing (Cole Partner Instrument Co., Chicago IL) and the sieving matrix loaded into the capillary from the syringe. The ends of the capillary were then immersed in reservoirs of IX TBE running buffer in a separation plate holder as described in Example 1. Detection was accomplished as described in Figure 8 with a stationary detection point 7.5 cm from the injection (cathode) end of the capillary. A 2 μL aliquot of the denatured, chilled DNA sequencing fragments was loaded into the injection well of the separation plate and electroinjeαion was run for 30 sec at 3.5 kV. The residual sample was removed from the injection well via flushing the well with - 1 mL of IX TBE run buffer. Electrophoresis was then conduαed for up to 35 minutes at 2 kV applied potential. Data collection was at 20 Hz on a Perkin Elmer/Nelson Model 1020 data system (PE/Nelson, Cupertino, CA).

Examples of the separation and analysis of the two ddTTP - terminated reaction niixture are shown in Figure 11. Figure 11A shows separation of the fragments in Tube A, which contained the ddTTP terminator from the start of the extension reaαion and thus contains fragments extending from the primer (18 bases) out to - 100 bases in length. Further exte ion/termination was not possible because the concentration of dNTP's in the reaction mixture was exhausted. Figure 11B shows the separation of the fragments in Tube B, which initially contained no ddTTP terminator and sufficient concentration of dNTP's to allow extension of the primer to - 100 bases before the fragments were again extended and terminated by addition of another limiting dose dNTP's/ddTTP.

The overlap region of the two sets of fragments generated in the set of two reactions is shown in detail in Figure 12A and 12B.