Sequencing large nucleic acid fragments

Abstract

The base sequence of large single stranded nucleic acids is determined by retarding the migration rate of a nucleic acid fragment of n bases below a migration rate which would otherwise be the same as the migration rate of a nucleic acid fragment of n bases in a polyacrylamide gel under continuous field gel electrophoresis. A plurality of sequences of electric field pulses is applied to the gel in one dimension.

Description

1. FIELD OF THE INVENTION

The present invention is directed to methods of sequencing single stranded nucleic acid molecules on polyacrylamide gels using gel electrophoresis and more particularly to such methods using pulsed field gel electrophoresis.

2. Background

Recently, advances in DNA and RNA research have led to many new and improved strategies for determining the nucleotide base sequence of these acids. In addition to cloning, polymerase chain reaction and enzymatic sequencing protocols, gel electrophoresis plays an important part in sequencing methods. This has created a demand for more rapid gel electrophoretic methods as well as methods having increased resolution. Unfortunately, gel electrophoretic techniques which use continuous electric fields up to at least 100 Volts per centimeter in a uniform gel (polyacrylamide) result in a loss of resolution on the gel between similar length fragments greater than about 300 bases. This is a severe limitation, increasing the number of subcloning steps as the gene must be cut many times into small fragments in order to determine the base sequence, and information about the structure of the entire gene is lost or becomes difficult to obtain.

Several attempts have been made to increase the band resolution of electrophoretic methods for separating large duplex DNA molecules in agarose gels. For example, U.S. Pat. No. 4,473,452 to Cantor et al. teaches the use of transverse electric fields which alternate between low and high intensities. This protocol allows the separation of larger size fragments of double stranded DNA in agarose gels at a higher speed and resolution. U.S. Pat. No. 4,740,283 to Laas et al. teaches a pulsed field gradient gel electrophoretic apparatus wherein the electrodes are oriented to provide a three-dimensional field across the face of the gel rather than in the plane of the gel. As a result, the molecules from the gel proceed down their respective lanes in a sawtooth matter. Both the '452 and '283 electrophoretic methods are inconvenient because they cannot be run on conventional electrophoretic equipment due to the requirement of special electrode configurations. Moreover, their use in sequencing protocols is unclear.

U.S. Pat. No. 4,737,251 to Carle et al. teaches an electrophoretic method for separating large molecular weight DNAs in agarose gel wherein the electric field is periodically inverted essentially in one dimension. A higher voltage or longer time is used in one direction than in the other. This method is thus used for separating large molecular weight DNAs wherein exceptional resolution is not necessary, using time intervals for field duration which range from seconds to hours.

In an effort to increase resolution in gel electrophoresis for sequencing single stranded DNA molecules, Tokita et al. in U.S. Pat. No. 4,904,366 teach lowering the ionic strength of the buffer solution in the polyacrylamide gel near the detector on a DNA base sequencer. The electric field intensity near the detector is thus increased, resulting in a higher migration speed and enhancing the resolving power of the apparatus. The loss of band resolution between large single stranded DNA fragments is thought to be caused by a phenomenon relating to the alignment of large molecules (larger than gel pore size) in the electric field which affects the migration patterns of the large sized fragments in the polyacrylamide gel rather than being directly related to the pH of the gel.

In a constant electric field, nucleic acid fragments containing up to about 300 bases migrate at a rate which is inversely proportional to their base content number, i.e., a fragment of n+1 base length migrates slower than one which is n bases long, which in turn migrates slower than one of n-1 bases, etc. As a result of this phenomenon, the bands of nucleic acid on a polyacrylamide sequencing gel are "compressed", having no discernable resolution between nucleic acid fragments having successively longer base lengths. In automated sequencers, currently being used in the Human Genome program, the bands which indicate the arrival of a particular base at the bottom of a gel are broadened for a number of reasons, resulting in poor resolution between them on a chart recorder. Referring to FIG. 1 a schematic of a chart recorder printout, bands 30, 31, 32, 33 and 34, corresponding to the same number of bases in a nucleic acid fragment are easily resolved. In contrast, bands 400, 401, 402, 403, and 404 show very poor resolution between them. Prior art approaches to overcoming this limitation of resolution have involved the use of pulsed fields with limited success.

For example, Lai et al. in "Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels", Electrophoresis, 10, 65-70, 1989, discuss the application of field inversion gel electrophoresis to single stranded DNA molecules having a base length greater than 130. The migration rate of DNA molecules larger than 130 bases was shown to be retarded as compared to their migration rates in conventional and unidirectional pulsed gels. The application of the Lai et al. method, however, failed to retard the migration of nucleic acid fragments larger than 600 bases long. While retarding the migration rate of fragments 130 bases long is certainly of interest, it is, however, somewhat inconsequential. At a length of 130 bases, using conventional electrophoretic techniques, nucleic acid fragments migrate through the gel at a rate which is inversely proportional to their base length and hence produce satisfactory band resolution on the gel. Therefore, methods which resolve single stranded DNA fragments of higher base lengths, the fragments of which would otherwise migrate at a rate the same as a fragment using constant field electrophoresis, are still needed.

SUMMARY OF THE INVENTION

The present invention pertains to a method of determining the base sequence of large single stranded nucleic acid fragments using gel electrophoresis. The method comprises retarding the migration rate of a nucleic acid fragment of n bases below a migration rate which would otherwise be the same as the migration rate of a nucleic acid fragment of n bases in a polyacrylamide gel under continuous field gel electrophoresis. A plurality of sequences of electric field pulses is applied in one dimension to the gel. Each of the sequences is comprised of a first pulse of a positive magnitude, applied for a first time period, and a second pulse of a negative magnitude applied for a second time period. The method is especially useful in sequencing long nucleic acid fragments, especially those which are longer than 300 bases and preferably longer than 600 bases.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention can be obtained by reference to the accompanying drawings wherein:

FIG. 1 shows schematic fluorescence data obtained on an automatic sequencer for relatively low fragment lengths, and broadening for higher fragment lengths using prior art methods.

FIG. 2 shows a comparison between the prior art and the present invention for schematic fluorescence data obtained for higher fragment lengths.

FIG. 3 shows the effect of the ionic strength (TBE conc.) of the gel on the relative electrophoretic mobility of DNA fragments (1X TBE, .smallcircle.; 0.5X ; 1.5X TBE, ).

FIG. 4 shows the effect of the polyacrylamide gel concentration; (4%, .smallcircle.; 6%, ; 8%; ; 12%, ) on the relative mobility of single stranded DNA fragments ranging in length from 80 to 755 bases at a constant of 2000 Volts.

FIG. 5 shows the effect of temperature on the relative electrophoretic mobility of DNA fragments. (55.degree. C., ; 45.degree. C., ; 40.degree. C., ; 35.degree. C., ; 30.degree. C., ,)

FIG. 6 shows the effect of field strength on the relative electrophoretic mobility of DNA fragments. (750V, +; 1000V, .smallcircle.; 1500V, ; 2000V, ; 2500V; ; 3000V, ; 4000V, ; 5000V )

FIG. 7 shows the effect of intermittent field pulsing on the relative electrophoretic mobility of single stranded DNA fragments compared to the mobility with a continuous field of the same intensity. (1000V, +; 1s, 1000V and 2s, 0V, .smallcircle.; 1s, 1000V and 5s, 0V, )

FIG. 8 shows the effect of pulse sequences according to an embodiment of the present invention compared to a continuous field of the same intensity on the relative electrophoretic mobility of DNA fragments. (2000V +; 1 ms, 2000V and 1ms, -300V, .smallcircle.; 1ms, 2000V and 1ms, -620V, .smallcircle.; 2ms, 2000V and 4ms, -630V, .smallcircle.; 2ms, 2000V and 2ms, -1000V ; 2ms, 2000V, 1ms, -200V, )

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In preparation for the method of the present invention, single stranded nucleic acids, as for example, DNA, can be prepared for sequencing prior to electrophoresis by any of the methods known in the art, for example, the Maxam and Gilbert chemical method or the Sanger, Nicklaus and Coulsen enzymatic method (Sambrook, Maniatis and Frisch, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989). An electrophoretic gel is prepared from polyacrylamide or an equivalent gel and the nucleic acid fragments are deposited on the gel in a conventional manner (e.g., 4 wells at the upper edge of the gel, each corresponding to one of the 4 bases). An automated sequencer (e.g., Pharmacia, duPont, Applied Systems) in which the flourescently labeled DNA fragments migrating down the gel are detected at the bottom by emitting a signal which is recorded on a chart recorder can be used. Alternatively, the method of the present invention can be used with more conventional sequencing methods wherein the sequence of the DNA is read directly off of the gel. Both methods will be immediately recognized by those skilled in the art.

By applying a plurality of sequences of electric field pulses in one dimension to the gel, the migration rate of nucleic acid fragments, as for example DNA and RNA fragments, containing n bases is retarded below a rate which would otherwise be the same as the migration rate of a nucleic acid fragment of n bases in a polyacrylamide gel under a constant electric field. Although the details of the molecular mechanism which governs the retardation of migration rates is quite complicated, in summary, during the time involved in which the negative pulse is applied, there is a backward displacement of the fragments on the gel. If the time period is long enough to allow a fragment of n bases to relax to the negative field pulse, the net displacement of the fragment with n+1 bases is less than, and hence its migration rate retarded below, the net displacement of a fragment with n bases. Each of the sequences is comprised of a first pulse of a positive magnitude which is applied for a given time, and a second pulse of a negative magnitude which is applied for a given time.

The present invention, due to its migration retarding effect, is therefore useful in sequencing nucleic acid fragments having a length greater than 300 bases, preferably greater than 400 bases and most preferably greater than 600 bases. To sequence base lengths lower than this, conventional constant field electrophoresis is usually quite effective. Thus a constant field may be applied until the sequencing reaches these ranges, at which time application of a plurality of sequences of electric pulses to the gel is employed. There is theoretically no limit to the upper number of bases between which the present invention can resolve and hence sequence. However, present sequencing methods can only synthesize fragments of nucleic acids up to about 2Kb using available polymerases. More polymerases will be needed before sequencing can approach these ranges, but upon their development the present invention would permit resolution of fragments of such lengths.

The retarded migration rates result in bands of nucleic acid fragments on the gel being "decompressed", thereby increasing the number of readable DNA fragments and hence readable bases per gel. With an automated sequencer the retarded migration rate results in bands on the chart recorder being spatially separated so as to facilitate nucleic acid base sequence reading. Reference is made to FIG. 2 wherein improved resolution between bands on a chart recorder is illustrated as a result of the present invention. Chart recorder sequence data for constant field electrophoresis is shown on the top line for nucleic acid fragments 400-404 bases in length. Resolution between bands of successively higher base length are poorly resolved because of the width of the bands. In contrast, the bottom line shows markedly increased resolution between nucleic acid fragments of 400 and 401 bases in length when the methods of the present invention are employed. The improved resolution is a direct result of the retarded migration rates obtained with the present invention. Essentially, the lower the time interval between successive signal detections on the recorder.

According to the present invention, the duration of at least one of the first time period (T.sub.1) and the second time period (T.sub.2) is from about 100 microseconds to about 10 milliseconds, preferably from about 1 millisecond to about 5 milliseconds and more preferably from about 2 milliseconds to about 4 milliseconds. The absolute value of at least one of the first voltage (V.sub.1) and the second voltage (V.sub.2) is greater than zero and less than 10,000 Volts, preferably from about 300 to about 5000 Volts. In a preferred embodiment, the absolute value of the first voltage V.sub.1 is greater than or equal to the absolute value of the second voltage V.sub.2.

In one preferred embodiment, the value of the product of the first time period and the first voltage of a positive magnitude plus the value of the product of the second time period and the second voltage of negative magnitude divided by the sum of the first and the second time periods is essentially zero. Essentially zero is intended to mean that the average electric field as applied throughout the duration of the sequence is at or near zero. For example, if a sequence was comprised of a first pulse applied at +2000V for 1 millisecond and a second pulse was applied at -1000V for 2 milliseconds, the average electric field would be zero. Alternatively, if the value of the product of the first time period and the first voltage of a positive magnitude plus the value of the product of the second time period and the second voltage of negative magnitude divided by the sum of the first and the second time periods is less than about 50% of the larger of the first and second voltages, it can be considered to be essentially zero.

A DC power source having an available voltage range of +10,000 to -10,000 Volts and a slew rate of 200-500 Volts/microsec. may be used to provide the sequences of electric field pulses to the gel. Slew rate is used according to its known meaning in the art and is defined as the rate of voltage change/time or dV/dt. A timer/switcher capable of switching voltage in the order of microseconds should be used to control the power source.

In further embodiments, the method according to the present invention further comprises another step of repeating the plurality of sequences or a method wherein at least one of the first pulse and the second pulse in the sequence of pulses is comprised of a number of subpulses. Subpulses are pulses of a reverse voltage magnitude or zero which go back to the original magnitude at least once before the application of another subpulse of reverse magnitude. The duration of the inversion is in the order of microseconds. Subpulses are responsible for what is referred to as a shaking effect, helping the nucleic acid fragments move through the pores of the gel.

The following examples are illustrative in nature and are not intended to limit the scope of the invention in any way. Other equivalent methods of practicing the present invention may occur to those skilled in the art upon reading the present specification.

EXAMPLES

Methods and Materials

Gel solutions are prepared by mixing together 7M urea (21g, ICN Biochemicals), acrylamide in a stock solution of acrylamide-N,N-methylene-bis-acrylamide (38:2 Broad) to a final acrylamide concentration of 4%, 6%, 8% and 12% respectively, and water to a total volume of 50 ml. TBE buffer (0.9 molar trisbase, 0.9 molar boric acid, 20 millimolar EDTA) is added to a final concentration of buffer between 0.5 and 1.5X using a stock solution 10X concentrated. Ammonium persulfate (0.07 to 0.8% weight volume) and N,N,N,tetra-methylethylene diamine (TEMED) (0.087 to 0.04% weight per volume) are added, amount dependent on the acrylamide concentration.

The gel solution is poured between a glass plate and a thermostatic plate (Pharmacia LKB) (550 millimeters by 220 millimeters) separated by 0.2 millimeters. The two plates are treated respectively by binding a repellant silane (Pharmacia LKB). The gel solution is allowed to polymerize for thirty minutes. Electrophoresis is carried out with the 2010 MACRO FOUR unit (Pharmacia LKB) using the same electrode buffer concentration as in the gel. Each gel is prerun at 2000 Volts constant voltage for two hours at 50.degree. C. to reach a constant current plateau value. DNA from the bacterial phage M13 MP18 (Pharmacia LKB) is used as a standard marker and the radio labeled .sup.35 S product of the dideoxy sequencing reaction using the T7 bacterial phage DNA fragments is prepared using a standard sequencing kit (Pharmacia LKB).

Relative mobility of the DNA fragments is calculated according to the distance in millimeters that a fragment of a given length migrated down the gel in relation to a fragment of 80, 90 or 100 bases long according to the formula: ##EQU1##

The relative mobilities, as well as the actual distances in millimeters traveled by the fragments on the gel are reported in tables I-X corresponding to examples 1-10 below. Reported voltages are applied across the entire length of the gel (550 mm).

Comparative Example 1

Electrophoresis is run at a constant electric field of 2000V in a 6% polyacrylamide gel. The ionic strength of the gel is varied from 0.5X to 1.5X TBE. The run is stopped when the dye marker control xylene cyanol reached the bottom of the gel. Relative mobilities of DNA fragments as compared to an 80 base fragment are recorded and are reported in Table I. The overall effect of varying the ionic strength on the relative mobilities of the fragments is seen in FIG. 3.

Comparative Example 2

Electrophoresis is run at a constant electric field of 2000V and a constant ionic strength of 1X TBE in 4%, 6%, 8% and 12% acrylamide gels. The experiment is stopped when the dye marker control (xylene cyanol) reaches the bottom of the gel. The relative mobility of the fragments compared to an 80 base fragment are reported in Table II. The effect of acrylamide concentration is illustrated in FIG. 4.

Comparative Example 3

Electrophoresis is run at a constant electric field of 2000V in 6% acrylamide gel at ionic strength of IX TBE at the temperatures of 55.degree. C., 45.degree. C., 40.degree. C., 35.degree. C. and 30.degree. C.. Relative mobility compared to an 80 base fragment is reported in Table III. The effect of temperature variation on the relative mobility is illustrated in FIG. 5.

Comparative Example 4

Electrophoresis is run in 6% acrylamide gel at an ionic strength of 1X TBE, at a temperature of 50.degree. C. at constant electric field strengths of 750 Volts, 1000 Volts, 1500 Volts, 2000 Volts, 2500 Volts, 3000 Volts, 4000 Volts and 5000 Volts. The relative mobility of the fragments as compared to a 90 base fragment is reported in Table IV. The effect of field strength on the relative mobilities of the fragments is illustrated in FIG. 6.

Comparative Example 5

Electrophoresis is run in 6% acrylamide gel at an ionic strength of 1X TBE and a temperature of 50.degree. C. The buffer is changed daily. A constant electric field of 1000 Volts is applied for eight hours, followed by a sequence of pulses of 1 second at 1000 Volts and 2 seconds at zero Volts, followed by a sequence of 1 second at 1000 Volts and 5 seconds at zero Volts, followed by 1 seconds at 1000 Volts and 1 seconds at zero Volts. The relative mobility of the fragments as compared to an 80 base length fragment is reported in Table V. The effect of this "intermittent" field on the relative mobilities is illustrated by FIG. 7.

Example 6

Electrophoresis is run in 6% acrylamide gel having an ionic strength of 1X TBE at a temperature of 50.degree. C. A continuous electric field of 2000 Volts is run for 4.4 hours. This is followed in several runs using the following pulsed sequences: sequence of 1 millisecond at 2000 Volts and 1 millisecond at -300 Volts; a sequence of 1 millisecond at 2000 Volts and 1 millisecond at -620 Volts; a sequence of 2 milliseconds at 2000 Volts and 4 milliseconds at -630 Volts; a sequence of 2 milliseconds at 2000 Volts and 2 milliseconds at -1000 Volts; a sequence of 2 milliseconds at 2000 Volts and 1 millisecond at -2000 Volts. The relative mobility of the fragments to a 100 base length fragment is reported in Table VI. FIG. 8 illustrates the effect of these pulsed sequences on the relative mobility of the fragments.

Example 7

Electrophoresis is run in 6% acrylamide gel having an ionic strength of 1X TBE at a temperature of 50.degree. C. A continuous electric field of 2000 Volts is applied for 4.4 hours. This is followed by a sequence of pulses of millisecond at 2000 Volts and 1 millisecond at -300 Volts; a sequence of 2 milliseconds at 2000 Volts; and 2 milliseconds at -300 Volts; a sequence of 2 milliseconds at 2000 Volts and 4 milliseconds at -300 Volts. The relative mobility of the fragments to a 100 base length fragment is reported in Table VII.

Example 8

Electrophoresis is run in 6% acrylamide gel having an ionic strength of 1X TBE at a temperature of 50.degree. C. A continuous electric field of 2000 Volts is applied for 4 hours. This is followed in several runs using the following pulse sequences: sequence of 1 second at 2000 Volts alternating with 1 second at -620 Volts; a sequence of 10 milliseconds at 2000 Volts alternating with 10 milliseconds at -620 Volts; a sequence of 5 millisecond at 2000 Volts alternating with 5 milliseconds at -620 Volts; a sequence of 2 millisecond at 2000 Volts alternating with 2 milliseconds at -620 Volts; and finally a sequence of 1 millisecond at 2000 Volts alternating with 1 millisecond at -620 Volts. The relative mobility of the fragments as compared to 100 base length fragment is reported in Table VIII.

Example 9

Electrophoresis is run in 6% acrylamide gel having an ionic strength of 1X TBE at a temperature of 50.degree. C. A continuous electric field of 2000 Volts is applied for 4.4 hours. This is followed in several runs using the following pulse sequences: sequence of pulses of 2 milliseconds at 2000 Volts alternating with 1 millisecond at -620 Volts; a sequence of 2 milliseconds at 2000 Volts alternating with 2 milliseconds at -620 Volts; and a sequence of 2 millisecond at 2000 Volts alternating with 3 milliseconds at -620 Volts. The relative mobility of the fragments as compared to a 100 base fragment is reported in Table IX.

Example 10

Electrophoresis is run in 6% acrylamide gel having an ionic strength of 1X TBE at a temperature of 50.degree. C. A continuous field of 2000 Volts is applied for 4.4 hours, followed by a pulsed field sequence of 1 millisecond at 2000 Volts alternating with 4 milliseconds at -630 Volts run for 40.8 hours. Relative mobility of the fragments to a 100 base fragment is reported in Table X.

TABLE I__________________________________________________________________________Base Distance (mm) Relative Distance (mm) Relative Distance (mm) RelativeNumber 1X TBE Mobility 1X TBE 0.5X TBE Mobility 0.5X TBE 1.5X TBE Mobility 1.5X__________________________________________________________________________ TBE755 97 0.191 111 0.218 81 0.159720 100 0.197 113 0.222 83 8.163680 103 0.203 118 0.228 86 9.169454 131 0.258 143 0.281 119 0.233425 139 0.274 149 0.293 126 0.247401 144 0.284 156 9.306 132 0.259371 153 0.302 165 0.324 141 0.278350 161 0.318 173 0.340 149 0.292335 167 0.329 179 0.352 156 0.306300 184 0.363 97 0.387 173 0.339260 210 0.414 221 0.434 200 0.392250 217 0.428 228 0.448 208 0.408240 226 0.446 236 0.464 216 0.424230 234 0.462 244 0.479 225 0.441220 241 0.475 252 0.495 235 0.461210 252 0.497 262 0.515 244 0.478200 261 0.515 272 0.534 255 0.500190 273 0.538 284 0.558 267 0.524180 288 0.568 298 0.585 281 0.561170 302 0.596 311 0.611 295 0.578160 316 0.623 325 0.639 310 0.608150 332 0.655 341 0.670 327 0.641140 348 0.586 357 0.701 344 0.675130 367 0.724 376 0.739 365 0.716120 387 0.763 98 0.782 389 0.763110 408 0.806 420 0.825 414 0.812100 435 0.858 449 0.882 445 0.873 90 467 0.921 481 0.945 480 0.941 80 507 1.000 509 1.000 510 1.000__________________________________________________________________________ TABLE II__________________________________________________________________________ RelativeBase Distance (mm) Distance (mm) Distance (mm) Distance (mm) Mobility Relative Relative RelativeNumber 4% gel 6% gel 8% gel 12% gel 4% Mobility 6% Mobility Mobility__________________________________________________________________________ 12%755 96 102 86 86 0.217 0.198 0.193 0.172720 100 104 89 88 0.226 0.202 0.200 0.176680 103 107 90 91 0.233 0.207 0.202 0.182454 140 137 118 118 0.316 0.208 0.261 0.236425 147 145 121 124 0.332 0.281 0.272 0.248401 154 151 127 129 0.348 0.283 0.285 0.258371 165 161 135 138 0.372 0.312 0.303 0.276350 174 169 142 146 0.393 0.328 0.319 0.292335 181 176 147 151 0.400 0.341 0.330 0.302300 200 194 163 168 0.451 0.376 0.366 0.336260 224 221 182 191 0.506 0.428 0.409 0.382250 231 228 169 198 0.521 0.442 0.425 0.396240 239 236 195 206 0.540 0.457 0.438 0.412230 248 244 202 214 0.560 0.473 0.454 0.428220 255 253 204 222 0.576 0.490 0.458 0.444210 263 264 217 231 0.594 0.512 0.468 0.462200 272 275 227 241 0.614 0.533 0.510 0.482190 283 287 237 252 0.639 0.558 0.533 0.504180 294 301 248 265 0.664 0.583 0.557 0.530170 303 315 260 278 0.684 0.610 0.584 0.556160 311 326 274 293 0.702 0.638 0.616 0.586150 326 344 289 310 0.740 0.667 0.649 0.620140 341 362 305 328 0.770 0.702 0.685 0.656130 355 382 322 348 0.801 0.749 0.724 0.696120 370 405 342 371 0.835 0.785 0.769 0.742110 385 427 363 397 0.869 0.828 0.816 0.794100 402 457 367 425 0.907 0.886 0.870 0.850 90 410 483 414 459 0.944 0.936 0.930 0.918 80 443 516 445 500 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE III__________________________________________________________________________ Distance Relative Distance Relative Distance Relative Relative RelativeBase (mm) Mobility (mm) Mobility (mm) Mobility Distance (mm) Mobility Distance MobilityNumber 56.degree. C. 56.degree. C. 45.degree. C. 45.degree. C. 40.degree. C. 40.degree. C. 35.degree. C. 35.degree. C. 30.degree. C. 30.degree.__________________________________________________________________________ C.755 96 0.191 95 0.187 100 0.196 93 0.185 94 0.195720 97 0.183 98 0.193 103 0.202 95 0.188 97 0.201680 101 0.201 101 0.199 107 0.210 98 0.194 100 0.207454 133 0.265 132 0.260 143 0.281 131 0.260 139 0.288425 140 0.279 138 0.272 150 0.295 137 0.272 148 0.307401 147 0.293 144 0.283 158 0.310 143 0.284 155 0.322371 156 0.311 153 0.301 189 0.332 154 0.308 165 0.342350 165 0.829 161 0.317 178 0.350 160 0.317 173 0.359335 171 0.341 167 0.329 184 0.361 166 0.320 179 0.371300 188 0.375 184 0.362 202 0.397 183 0.363 195 0.405260 211 0.420 210 0.413 228 0.448 209 0.415 220 0.458250 219 0.436 216 0.425 235 0.462 215 0.427 228 0.469240 228 0.450 225 0.443 240 0.472 223 0.442 235 0.488230 234 0.466 233 0.459 254 0.499 231 0.458 243 0.504220 244 0.486 241 0.474 262 0.515 241 0.471 253 0.525210 253 0.504 250 0.492 273 0.536 249 0.494 262 0.544200 263 0.524 261 0.514 282 0.554 260 0.516 272 0.564190 275 0.548 272 0.535 284 0.578 271 0.538 282 0.585180 288 0.574 285 0.581 305 0.599 284 0.583 292 0.606170 300 0.598 299 0.589 317 0.623 296 0.591 303 0.629160 315 0.827 313 0.616 329 0.646 312 0.619 315 0.654150 332 0.661 329 0.648 343 0.674 328 0.651 327 0.678140 350 0.497 345 0.679 360 0.747 345 0.685 341 0.707130 379 0.737 363 0.715 380 0.747 363 0.720 358 0.743120 392 0.781 385 0.758 402 0.790 385 0.764 377 0.782110 415 0.827 409 0.805 427 0.839 409 0.812 393 0.828100 440 0.874 440 0.866 453 0.890 437 0.867 424 0.880 90 470 0.936 475 0.935 482 0.947 470 0.933 452 0.938 80 502 1.000 508 1.000 509 1.000 504 1.000 482 1.000__________________________________________________________________________ TABLE IV__________________________________________________________________________ Distance Distance Distance Distance Distance Distance Distance Distance DistanceBase (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm)Number 500 V 750 V 1000 V 1500 V 2000 V 2500 V 3000 V 4000 V 5000 V__________________________________________________________________________755 55 74 89 98 102 119 128 121 133720 58 77 92 101 104 121 131 124 136680 62 81 96 105 107 123 134 128 138590 74 93 106 116 117 133 144 135 146527 86 103 117 127 124 141 153 141 153454 102 119 132 144 137 154 169 153 168401 118 133 147 161 151 169 185 165 179271 130 143 158 172 169 179 195 173 188350 135 152 167 181 176 188 203 182 195325 147 158 173 188 194 194 210 188 202300 167 177 193 203 221 213 229 205 217260 196 204 220 236 228 237 255 227 240250 204 213 227 245 236 244 283 234 248240 212 220 235 254 244 252 270 242 253230 222 229 244 262 253 261 279 249 262220 232 238 253 271 264 272 290 258 270210 243 246 263 282 275 281 299 267 290200 255 258 276 292 287 203 309 278 291190 267 271 287 314 301 305 319 289 302180 280 284 300 327 315 318 331 302 315170 293 298 312 341 326 331 344 314 329160 307 313 327 355 344 346 359 328 342150 321 330 343 373 362 362 375 343 358140 337 347 359 394 382 380 394 359 374130 356 366 378 416 405 399 413 376 393120 378 388 400 441 427 424 437 398 418110 401 412 424 467 457 448 463 422 442100 429 439 453 496 438 477 490 450 470 90 461 466 482 507 519 482 503__________________________________________________________________________ Rel. Rel. Rel. Rel. Rel. Rel. Rel. Rel. Rel.Base Mob. Mob. Mob. Mob. Mob. Mob. Mob. Mob. Mob.Number 500 V 750 V 1000 V 1500 V 2000 V 2500 V 3000 V 4000 V 5000 V__________________________________________________________________________755 0.119 0.158 0.185 0.198 0.211 0.235 0.247 0.251 0.264720 0.126 0.165 0.191 0.204 0.215 0.239 0.252 0.257 0.268680 0.134 0.174 0.199 0.212 0.222 0.243 0.258 0.261 0.274590 0.161 0.200 0.220 0.234 0.242 0.262 0.277 0.280 0.290527 0.187 0.221 0.243 0.256 0.257 0.278 0.295 0.293 0.304454 0.221 0.255 0.274 0.290 0.284 0.304 0.326 0.317 0.330401 0.256 0.285 0.305 0.325 0.313 0.333 0.356 0.342 0.356271 0.282 0.307 0.328 0.347 0.333 0.353 0.376 0.359 0.374350 0.302 0.326 0.346 0.365 0.350 0.371 0.381 0.376 0.384325 0.319 0.339 0.359 0.379 0.364 0.383 0.405 0.390 0.402300 0.362 0.360 0.400 0.419 0.402 0.420 0.441 0.425 0.431260 0.425 0.438 0.456 0.476 0.458 0.467 0.491 0.471 0.477250 0.443 0.457 0.471 0.494 0.472 0.481 0.507 0.485 0.489240 0.460 0.472 0.488 0.512 0.489 0.497 0.520 0.502 0.503230 0.482 0.491 0.508 0.528 0.505 0.515 0.538 0.517 0.521220 0.503 0.511 0.525 0.546 0.524 0.536 0.559 0.535 0.537210 0.527 0.532 0.546 0.569 0.547 0.554 0.576 0.554 0.567200 0.553 0.554 0.568 0.589 0.569 0.578 0.595 0.577 0.579190 0.579 0.582 0.595 0.600 0.594 0.602 0.615 0.600 0.600180 0.607 0.609 0.622 0.633 0.623 0.627 0.638 0.627 0.626170 0.636 0.639 0.647 0.659 0.652 0.653 0.663 0.651 0.654160 0.666 0.672 0.678 0.688 0.679 0.882 0.692 0.680 0.680150 0.696 0.708 0.712 0.716 0.712 0.714 0.723 0.712 0.712140 0.731 0.745 0.745 0.752 0.749 0.750 0.759 0.745 0.744130 0.772 0.785 0.784 0.794 0.791 0.787 0.798 0.784 0.781120 0.820 0.833 0.830 0.839 0.839 0.836 0.842 0.826 0.827110 0.870 0.884 0.880 0.889 0.884 0.894 0.892 0.876 0.879100 0.931 0.942 0.948 0.942 0.946 0.941 0.944 0.934 0.934 90 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE V__________________________________________________________________________ Constant Field Relative Relative Relative Constant Field Distance (mm) Distance (mm) Relative Mobility Mobility MobilityBase Distance (mm) Distance (mm) 1 s 1000 V; 1 s 1000 V; Mobility 1 s 1000 V; 1 s 1000 1 s 1000 V;Number 2000 V 1 s 1000 V; 1 s OV 2 s OV 5 s OV 2000 V 1 s OV 2 s OV 5 s__________________________________________________________________________ OV755 66 78 62 55 0.136 0.153 0.129 0.115720 69 81 65 57 0.142 0.159 0.135 0.119680 73 85 69 59 0.150 0.167 0.143 0.123590 83 97 79 67 0.170 0.190 0.163 0.141527 94 109 90 76 0.193 0.214 0.187 0.159454 109 127 108 93 0.224 0.249 0.224 0.195425 117 135 117 101 0.240 0.265 0.248 0.211401 124 143 125 109 0.255 0.280 0.258 0.228371 134 155 137 119 0.275 0.304 0.284 0.249350 142 164 146 128 0.292 0.322 0.303 0.268335 140 170 153 134 0.306 0.333 0.317 0.280300 167 189 172 154 0.343 0.371 0.357 0.322260 194 215 200 181 0.398 0.422 0.415 0.379250 202 223 207 189 0.415 0.437 0.428 0.395240 210 232 212 197 0.431 0.455 0.440 0.412230 219 241 226 207 0.450 0.473 0.489 0.433220 229 251 236 218 0.470 0.492 0.498 0.456210 238 261 246 227 0.489 0.512 0.518 0.475200 249 272 257 240 0.511 0.533 0.533 0.502190 261 284 268 254 0.536 0.557 0.556 0.531180 275 297 281 266 0.565 0.582 0.583 0.558170 289 310 294 280 0.593 0.608 0.610 0.586160 304 326 305 295 0.624 0.639 0.633 0.617150 320 343 320 309 0.657 0.673 0.664 0.646140 338 361 335 323 0.694 0.708 0.695 0.676130 358 381 351 341 0.735 0.747 0.728 0.713120 379 402 372 362 0.778 0.788 0.772 0.757110 401 425 394 384 0.823 0.833 0.817 0.803100 426 451 419 412 0.875 0.884 0.868 0.862 90 455 483 450 442 0.934 0.941 0.934 0.925 80 487 510 482 478 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE VI__________________________________________________________________________ Constant Field Relative Constant Field Distance (mm) Distance (mm) Distance (mm) Distance (mm) Distance MobilityBase Distance (mm) 1 s 2 KV; 1 s 2 KV; 1 s 2 KV; 2 s 2 KV; 2 s KV; 2000 V/Number 2000 V/4.4 hrs. 1 s -300 V 1 s -620 V 4 s -630 V 2 s -1000 V 1 s -2 kV 4.4__________________________________________________________________________ hrs.755 110 73 70 68 73 80 0.225720 113 76 74 72 79 86 0.232680 117 80 78 77 83 91 0.240590 127 90 92 94 98 106 0.260527 136 98 104 108 111 121 0.279454 154 113 119 131 132 142 0.316425 162 120 127 141 142 152 0.332401 170 127 136 151 152 162 0.348371 182 136 144 164 162 176 0.373350 190 144 155 174 174 185 0.389335 197 149 141 183 182 194 0.404300 217 166 182 204 204 216 0.445260 247 191 211 238 235 249 0.506250 254 198 219 246 243 259 0.520240 263 206 228 256 252 266 0.539230 273 213 237 267 262 278 0.559220 282 222 248 277 273 288 0.578210 292 230 257 288 283 298 0.598200 302 240 268 299 294 309 0.619190 314 250 282 310 305 321 0.643180 326 260 296 323 316 334 0.672170 341 273 312 337 331 347 0.699160 355 287 327 351 344 364 0.727150 374 302 344 370 361 383 0.766140 394 317 362 390 380 404 0.807130 417 335 381 415 402 427 0.855120 441 353 404 442 426 455 0.904110 485 371 430 470 453 481 0.953100 488 393 461 490 481 507 1.000__________________________________________________________________________ Relative Relative Relative Relative Relative Mobility Mobility Mobility Mobility Mobility Base 1 s 2 KV; 1 s 2 KV; 1 s 2 KV; 2 s 2 2 s KV; Number 1 s - 300 V 1 s -620 V 4 s -630 V 2 s -1000 1 s -2__________________________________________________________________________ KV 755 0.166 0.152 0.136 0.152 0.158 720 0.193 0.161 0.144 0.164 0.170 680 0.204 0.169 0.154 0.173 0.179 590 0.226 0.200 0.188 0.204 0.209 527 0.249 0.226 0.216 0.231 0.239 454 0.288 0.258 0.263 0.274 0.280 425 0.305 0.275 0.283 0.295 0.300 401 0.323 0.293 0.303 0.316 0.320 371 0.346 0.312 0.329 0.337 0.347 350 0.368 0.336 0.349 0.362 0.365 335 0.379 0.349 0.367 0.378 0.383 300 0.422 0.395 0.409 0.424 0.426 260 0.486 0.458 0.477 0.489 0.491 250 0.504 0.475 0.493 0.505 0.511 240 0.524 0.495 0.513 0.524 0.529 230 0.542 0.514 0.535 0.545 0.548 220 0.585 0.536 0.555 0.568 0.568 210 0.585 0.557 0.577 0.586 0.588 200 0.611 0.581 0.599 0.611 0.609 190 0.636 0.612 0.621 0.634 0.633 180 0.662 0.642 0.647 0.661 0.650 170 0.695 0.677 0.675 0.686 0.684 160 0.730 0.709 0.703 0.715 0.718 150 0.768 0.746 0.741 0.751 0.755 140 0.807 0.785 0.782 0.790 0.797 130 0.862 0.826 0.832 0.836 0.842 120 0.897 0.876 0.886 0.886 0.897 110 0.944 0.933 0.942 0.942 0.949 100 1.000 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE VII__________________________________________________________________________ Constant Constant Field Relative Relative Relative Field Distance (mm) Distance (mm) Distance (mm) Relative Mobility Mobility MobilityBase Distance (mm) 1 ms 2 KV; 2 ms 2 KV; 2 ms 2KV; Mobility 1 ms 2 KV; 2 ms 2 2 ms 2 KV;Number 2000 V 1 ms -300 V 2 ms -300 V 4 ms -300 V 2000 V 1 ms -300 V 2 ms -300 4 ms -300__________________________________________________________________________ V755 110 73 82 80 0.225 0.166 0.176 0.172720 113 76 86 85 0.232 0.193 0.184 0.182680 117 80 91 89 0.240 0.204 0.195 0.191590 127 90 106 103 0.260 0.229 0.227 0.221527 136 93 118 117 0.279 0.249 0.263 0.251454 154 113 137 136 0.316 0.288 0.293 0.292425 162 120 148 146 0.332 0.305 0.313 0.313401 170 127 155 155 0.348 0.323 0.332 0.333371 182 136 164 169 0.373 0.346 0.351 0.363350 190 144 177 177 0.389 0.366 0.379 0.380335 192 149 184 185 0.393 0.379 0.394 0.397300 217 166 203 206 0.445 0.422 0.435 0.442260 247 191 233 235 0.506 0.466 0.498 0.504250 254 198 241 242 0.520 0.504 0.516 0.519240 263 206 250 251 0.539 0.524 0.535 0.539230 273 213 260 261 0.559 0.542 0.557 0.560220 282 222 270 270 0.578 0.565 0.576 0.579210 292 230 280 280 0.598 0.585 0.600 0.601200 302 240 290 291 0.619 0.611 0.621 0.624190 314 250 301 302 0.643 0.636 0.645 0.648180 326 260 314 314 0.672 0.662 0.672 0.674170 341 273 326 326 0.699 0.695 0.698 0.700180 355 287 338 339 0.727 0.730 0.724 0.727150 374 302 354 354 0.766 0.768 0.758 0.760140 394 317 372 371 0.807 0.807 0.797 0.796130 417 335 394 392 0.855 0.852 0.844 0.841120 441 353 417 415 0.904 0.898 0.893 0.891110 465 371 440 440 0.953 0.944 0.942 0.944100 483 393 467 466 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE VIII__________________________________________________________________________ Constant Field Constant Field Distance (mm) Distance (mm) Distance (mm) Distance (mm) Distance RelativeBase Distance (mm) 1 s 2 KV; 10 ms 2 KV; 5 ms 2 KV; 2 ms 2 KV; 1 ms 2 KV; MobilityNumber 2000 V/4 hrs. 1 s -620 V 10 ms -620 V 5 ms -620 V 2 ms -620 V 1 ms -620 2000 V/4__________________________________________________________________________ hrs.755 104 116 114 90 84 70 0.240720 106 122 118 94 89 74 0.245680 109 124 121 99 93 78 0.252590 118 133 131 112 107 92 0.273527 127 142 142 123 119 104 0.293454 143 158 159 141 138 119 0.330425 150 165 166 150 146 127 0.346401 158 174 174 158 155 135 0.365371 167 186 185 171 167 144 0.386350 176 196 194 181 178 155 0.406335 182 203 201 189 186 161 0.420300 200 223 221 210 209 182 0.462260 225 253 251 239 239 211 0.520250 233 260 260 247 249 219 0.538240 241 271 270 257 257 228 0.557230 249 281 280 267 268 237 0.575220 259 293 290 278 280 246 0.598210 269 305 301 289 292 257 0.621200 280 318 314 301 305 268 0.647190 293 331 328 314 319 282 0.677180 305 345 344 328 333 296 0.704170 319 360 360 343 349 310 0.737160 334 377 376 358 365 323 0.771150 351 396 395 377 383 340 0.811140 369 416 415 396 403 358 0.852130 389 443 440 419 425 378 0.898120 410 470 466 449 454 400 0.947110 433 500 495 479 483 426 1.000__________________________________________________________________________ Relative Relative Relative Relative Relative Mobility Mobility Mobility Mobility Mobility Base 1 s 2 KV; 10 ms 2 KV; 5 ms 2 KV; 2 ms 2 1 ms 2 KV; Number 1 s -620 V 10 ms -620 V 5 ms -620 V 2 ms -620 1 ms -620__________________________________________________________________________ V 755 0.236 0.230 0.188 0.174 0.164 720 0.244 0.238 0.196 0.184 0.174 680 0.248 0.244 0.207 0.193 0.183 590 0.266 0.265 0.234 0.222 0.216 527 0.284 0.287 0.257 0.246 0.244 454 0.316 0.321 0.294 0.286 0.279 425 0.330 0.335 0.313 0.302 0.298 401 0.346 0.352 0.330 0.321 0.317 371 0.372 0.374 0.357 0.346 0.338 350 0.392 0.392 0.378 0.369 0.364 335 0.406 0.406 0.395 0.385 0.378 300 0.446 0.446 0.438 0.433 0.427 260 0.506 0.507 0.499 0.495 0.495 250 0.520 0.525 0.516 0.516 0.514 240 0.542 0.545 0.537 0.532 0.535 230 0.562 0.566 0.557 0.555 0.556 220 0.586 0.586 0.580 0.580 0.582 210 0.610 0.606 0.603 0.605 0.603 200 0.636 0.634 0.628 0.631 0.629 190 0.662 0.663 0.656 0.660 0.662 180 0.690 0.695 0.685 0.689 0.695 170 0.720 0.727 0.716 0.723 0.726 160 0.754 0.760 0.747 0.756 0.758 150 0.792 0.798 0.787 0.793 0.798 140 0.836 0.838 0.827 0.834 0.840 130 0.886 0.889 0.875 0.880 0.887 120 0.940 0.941 0.937 0.940 0.939 110 1.000 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE IX__________________________________________________________________________ Distance Distance Distance Constant Field Relative Relative Relative Constant Field (mm) (mm) (mm) Relative Mobility Mobility MobilityBase Distance (mm) 2 ms 2 KV; 2 ms 2 KV; 2 ms 2 KV; Mobility 2 ms 2 KV; 2 ms 2 2 ms 2 KV;Number 2000 V/4.4 hrs. 1 ms -620 V 2 ms -620 V 3 ms -620 V 2000 V/4.4 hrs. 1 ms -620 V 2 ms -620 3 ms -620__________________________________________________________________________ V755 110 86 84 73 0.225 0.183 0.165 0.151720 113 91 89 77 0.232 0.194 0.175 0.160680 117 94 93 82 0.240 0.200 0.183 0.170590 127 107 107 94 0.260 0.228 0.210 0.195527 136 119 118 107 0.279 0.253 0.232 0.222454 154 140 138 124 0.316 0.298 0.271 0.257425 162 149 146 133 0.332 0.317 0.287 0.276401 170 158 155 141 0.348 0.336 0.305 0.293371 182 169 167 154 0.373 0.360 0.328 0.320350 190 179 178 161 0.389 0.381 0.350 0.334335 192 186 196 169 0.393 0.396 0.365 0.351300 217 206 209 190 0.445 0.438 0.411 0.394260 247 234 239 218 0.506 0.498 0.470 0.452250 254 243 249 227 0.520 0.517 0.489 0.471240 263 251 257 236 0.539 0.534 0.505 0.490230 273 260 268 245 0.559 0.553 0.527 0.508220 282 271 280 255 0.578 0.577 0.550 0.529210 292 280 292 266 0.598 0.596 0.574 0.552200 302 292 305 279 0.619 0.621 0.599 0.579190 314 303 310 292 0.643 0.645 0.627 0.606180 326 311 333 306 0.672 0.642 0.654 0.635170 341 331 349 321 0.699 0.704 0.696 0.656160 355 341 365 338 0.727 0.726 0.717 0.701150 374 362 383 354 0.766 0.770 0.752 0.734140 394 380 403 373 0.807 0.809 0.792 0.774130 417 399 425 395 0.855 0.849 0.835 0.820120 441 420 454 420 0.904 0.894 0.892 0.871110 465 443 483 450 0.953 0.943 0.949 0.934100 488 470 509 482 1.000 1.000 1.000 1.000__________________________________________________________________________ TABLE X__________________________________________________________________________ Constant Field Constant FieldBase Distance (mm) Distance (mm) Relative Mobility Relative MobilityNumber 2000 V/4.4 hrs. 2 ms 2 KV; 4 ms -630 V 2000 V/4.4 hrs. 2 ms 2 KV; 4 ms -630__________________________________________________________________________ V755 110 68 0.225 0.136720 113 72 0.232 0.144600 117 77 0.240 0.154590 127 94 0.260 0.188527 136 108 0.279 0.216454 154 131 0.316 0.263425 162 141 0.332 0.283401 170 151 0.348 0.303371 182 164 0.373 0.329350 190 174 0.389 0.349335 196 183 0.402 0.367300 217 204 0.445 0.409260 247 238 0.506 0.477250 254 246 0.520 0.493240 263 256 0.539 0.513230 273 267 0.559 0.535220 282 277 0.578 0.555210 292 288 0.598 0.577200 302 299 0.619 0.599190 314 310 0.643 0.621180 328 323 0.672 0.647170 341 337 0.699 0.675160 355 351 0.727 0.703150 374 370 0.766 0.741148 394 390 0.807 0.782130 417 415 0.855 0.832120 441 442 0.904 0.886110 465 470 0.953 0.942100 488 499 1.000 1.000__________________________________________________________________________

Claims

1. A method of determining the base sequence of large single stranded nucleic acid fragments using gel electrophoresis, comprising:

applying to a polyacrylamide gell a mixture of single stranded nucleic acid fragments wherein the migration rate of a single stranded nucleic acid fragment of n+1 bases is substantially the same as the migration rate of a single stranded nucleic acid fragment of n bases in a polyacrylamide gel under a constant electric field;

retarding the migration create of the single stranded nucleic acid fragment of n+1 bases below the migration rate of the single stranded nucleic acid fragment of n bases by applying to the mixture of single stranded nucleic acid fragments a plurality of sequences of electric field pulses in one dimension to the gel, each of said sequences being comprises of a first pulse of a positive magnitude V.sub.1 applied for a time period of T.sub.1, and a second pulse of a negative magnitudeV.sub.2 applied for a time period T.sub.2, wherein t.sub.1 and V.sub.1 are not the same as T.sub.2 and V.sub.2 ; and sequencing the single stranded nucleic acid fragments.

2. The method according to claim 1, wherein said retarding of the migration rate comprises decompressing bands of single stranded nucleic acid fragments on the gel, thereby increasing the number of readable nucleic acid fragments on the gel.

3. The method according to claim 1, further comprising reading sequence data with an automated sequencer having a chart recorder, wherein bands on the chart recorder are spatially separated so as to facilitate single stranded nucleic acid base sequence determination.

4. The method according to claim 1, wherein at least one of T.sub.1 and T.sub.2 is from about 100 microseconds to about 10 milliseconds.

5. The method according to claim 4, wherein a least one of T.sub.1 and T.sub.2 is from about 1 millisecond to about 5 milliseconds.

6. The method according to claim 5, wherein at least one of T.sub.1 and T.sub.2 is from about 2 millisecond to about 4 milliseconds.

7. The method according to claim 1, wherein the absolute value of at least one of V.sub.1 and V.sub.2 is greater than zero and less than 10,000 Volts.

8. The method according to claim 7, wherein the absolute value of at least one of V.sub.1 and V.sub.2 is from about 300 to about 5000 Volts.

9. The method according to claim 8, wherein the absolute value of at least one of V.sub.1 and V.sub.2 is about 2000 Volts.

10. The method according to claim 1, wherein ##EQU2## is essentially zero.

11. The method according to claim 1, wherein the absolute value of V.sub.1 is greater than or equal to the absolute value of V.sub.2.

12. The method according to claim 11, wherein the absolute value of V.sub.1 is greater than the absolute value of V.sub.2.

13. The method of claim 1, wherein T.sub.1 is less than 2 milliseconds, T.sub.2 is less than 2 milliseconds, V.sub.1 is greater than 1500 Volts and less than 2500 Volts, and V.sub.2 is greater than -400 Volts and less than -200 Volts.

14. The method according to claim 1, wherein T.sub.1 less than 2 milliseconds, T.sub.2 is less than 2 milliseconds, V.sub.1 is greater than 1500 Volts and less than 2500 Volts, and is greater than -700 Volts and less than -600 Volts.

15. The method according to claim 1, wherein T.sub.1 is greater than 1 millisecond and less than 3 milliseconds, T.sub.2 is greater than 3 milliseconds and less than 5 milliseconds, V.sub.1 is greater than 1500 Volts and less than 2500 Volts, and V.sub.2 is greater than -700 Volts and less than -600 Volts.

16. The method according to claim 1, wherein T.sub.1 is greater than millisecond and less than 3 milliseconds, T.sub.2 is greater than 1 millisecond and less than 2 milliseconds, V.sub.1 is greater than 1500 Volts and less than 2500 Volts, and V.sub.2 is greater than -1500 Volts and less than -500 Volts.

17. The method according to claim 1, where T.sub.1 is greater than one millisecond and less than 3 milliseconds, T.sub.2 is less than 2 milliseconds, V.sub.1 is greater than 1500 Volts and less than 2500 Volts, and V.sub.2 is greater than -2500 Volts and less than -1500 Volts.

18. The method according to claim 1, wherein n is greater than 300.

19. The method according to claim 18, wherein n is greater than 400.

20. The method according to claim 19, wherein n is greater than 500.

21. The method according to claim 20, wherein n is greater than 600.

22. The method according to claim further comprising applying continuous field gel electrophoresis prior to the application of said a plurality of sequences of electric field pulses to the gel.

23. The method according to claim further comprising repeating the application of said plurality of sequences.

24. The method according to claim 1, wherein at least one of the first pulse and the second pulse is comprised of a plurality of subpulses.

25. A method of determining the base sequence of large single stranded ucleic acid fragments using gel electrophoresis, comprising:

applying to a mixture of single stranded nucleic acid fragments, wherein the migration rate of a single stranded nucleic acid fragment of n bases is substantially the same as the migration rate of a single stranded nucleic acid fragment of n+1 bases in a polyacrylamide gel under a constant electric field, a plurality of sequences of electric field pulses in one dimension to the gel, each of said sequences being comprises of a first pulse of positive magnitude V.sub.1 applied for a time period T.sub.1, and a second pulse of a negative magnitude V.sub.2 applied for a time period T.sub.2, wherein T.sub.1 and V.sub.1 are not the same as T.sub.2 and V.sub.2, and said T.sub.2 being of the appropriate duration to allow a fragment of n+1 bases to relax to the negative field pulse without allowing a fragment of n bases to relax to the negative field pulse thereby retarding the migration rate of a single stranded nucleic acid fragment of n+1 bases below the migration rate of a single stranded nucleic acid fragment of n bases.