Accelerated Articles Anal. Chem. 1995, 67, 1913-1919
High=SpeedDNA Sequencing by Using Mixed Poly(ethy1ene oxide) Solutions in Uncoated Capillary Columns Eliza N. Fung and Edward S. Yeung* Ames Laborator USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 5001 1
We describe a new non-cross-linked polymer matrix and a new column treatment procedure for high-speed DNA sequencing by using capillary electrophoresis. A solution of commercially available poly(ethy1ene oxide) (PEO) in two size ranges is used as the sieving matrix under denaturing conditions. When compared to a commercial 6% T polyacrylamide solution, the mixed PEO matrix offers identical separation performancefor the small DNA fragments, better performance for the large DNA fragments, increased separation speed, and a lower viscosity. To avoid degradation of the standard bonded coating on the capillary column over many runs, we evaluated the resolution of DNA fragments in the mixed PEO matrix in bare fused-silica capillaries. In a dry, fresh column, the performance is identical to that observed in coated (bonded) capillaries. To regenerate the favorable surface characteristics after each run, 0.1 N HCl was used to retitrate the surface silanol groups back to the fully protonated form. The sequencing runs in fact proceeded much faster, with bases 28-420 from the Sanger ladder eluting in a span of only 16 min. We observed little degradation of the performance over tens of sequencing runs.
Several research groups have shown that capillary gel electrophoresis (CGE) is an attractive alternative to slab gel electrophoresis (SGE) for DNA sequencing.’-’O The medium used (1) Cohen, A S.; Najarian, D. R.; Paulus, A; Guttman, A,; Smith, J. A; Karger. B. L. Proc. Natl. Acad. Sci. U S A . 1987,85, 9660-9663. (2) Drossman, H.; Luckey, J. A; Kostichka, A J.; D’Cunha. J.; Smith, L. M. Anal. Chem. 1990,62. 9W903. (3) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R; Grey, R; Wu, S.; Dovichi, N. J.; Fuller, C. Anal. Chem. 1991,63, 283552841,
0003-2700/95/0367-1913$9.00/0 0 1995 American Chemical Society
(cross-linked polyacrylamide), buffer composition, separation mechanism, sequencing chemistry, and tagging chemistry for CGE are all derived from proven SGE schemes. A 25fold increase in the sequencing rate per capillary (per lane) has already been demonstrated. This is a direct consequence of the small internal diameter of the capillary tubes, typically around 50-75 pm, greatly reducing Joule heating associated with the electrical current. Gel distortions and temperature gradients that can affect resolution of the bands are thus virtually absent. More importantly, much higher electric fields can be applied to speed up the separation. For comparison, conventional SGE is limited to field strengths below 50 V/cm while CGE has been successfully used for sequencing up to 500 V/cm.3 The unique aspect ratio of capillary gels (25-50 cm long) provides uniform field strengths, and the large surface-to-volumeratio favors efficient heat removal. These combine to produce much sharper bands than are possible in slab gels. Early on, cross-linked polymers such as (PA) were used as gel matrices in CGE because of their known utility in slab gels for the separation of proteins and DNA. The separation performance is controlled by the pore structure of the gel matrices, which is based on the amount and characteristics Chen, D. Y.;Swerdlow, H. P.; Harke, H. R.; Zhang, J. Z.; Dovichi, N. J. /. Chromatogr. 1991,559, 237-246. Baba, Y.;Tsuhako, M. Trends Anal. Chem. 1992,11, 280-287. Luckey, J. A; Drossman, H.; Kostichka, A J.; Mead, D. A; D’Cunha, J.; Noms, T. B.; Smith, L. M. Nucleic Acids Res. 1990,18, 4417-4421. Luckey, J. A; Smith, L. M. Anal. Chem. 1993,65, 2841-2850. Baba, Y.; Matsuura, T.;Wakamoto, IC;Morita, Y.; Nishitsu, Y. Anal. Chem. 1992,64, 1221-1225. Lu, H.; Aniaga, E.; Chen, D. Y.; Dovichi, N. J. J. Chromatogr., A 1994, 680, 497-501. Lu,H.; Aniaga, E.; Chen, D. Y.; Figeys, D.; Dovichi, N. J.J. Chromatogr., A 1994,680, 503-510.
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of the monomer and cross-linking agents. However,” due to the instability over time, irreproducibility in the polymerization processes, and the fragile nature of the medium, cross-linked PAS in CE have not been reported to last for more than a few runs and are therefore not suitable for large-scale DNA sequencing, especially in multiplexed operation. The longevity issue also applies to ultrathin gels.12-15 So, despite the distinct advantage of higher resolution for the large fragments in cross-linked PA, alternative sieving matrices are urgently needed. Low to moderate viscosity entangled polymers have been used to overcome some of the above problems.16-2j Unlike cross-linked gels, they are replaceable and more stable for use at higher temperatures and electric field strengths. Linear PA (0%0 has been used for the size separation of DNA or proteins by sieving.lbJi*6.27 In addition, methyl ~ e l l u l o s e , ~hydroxyalkyl ~J~ cellulose,2n-22 poly(hydroxymethacry1ate) and poly(ethy1ene glycolmethacrylate) ,23 and poly(viny1 a l ~ o h o l ) also * ~ ~have ~ ~ been employed for DNA separations. Several important problems remain before entangled polymers can be routinely used for large-scale DNA sequencing. Despite the grand idea of replacing the sieving matrix after every run, implementation has been difficult. For example,26the pressure required to replace the PA matrix (6%T,1 x TBE, 30%formamide, 3.5 M urea) in a 33-cm-long, 75-pm4.d. capillary in 3 min is 84 atm (1.25 x lo3 psi). Even more critical is that Pouiselle flow implies that the material near the capillary walls will require yet higher pressures or longer times to be cleaned out. The high pressure may preclude many simple, automated schemes for flushing out a large number of capillaries in an array. The preparation of the linear PA polymer solutions is also difficult to control and to reproduce. Only a few laboratories have repeated success. The polymerization process depends critically on oxygen content,**temperature,Z6 time for complete reaction, reagent purity, and contamination. While one day the Human Genome Project may drive commercial manufacturers to produce “standard” polymer mixtures, at the present time only a 10% T Swerdlow, H.: Dew-Jager, K E.: Brady. IC: Gray, R.; Dovichi, N. J.: Gesteland, R. Electrophoresis 1992,13, 475-483. Stegemann, J.: Schwager, C.; Erfle, H.: Hewitt, N.: Voss, H.: Zimmermann, J.; Ansorge, W. Nucleic Acids Res. 1991,19, 675-676. Brumley, R. L.: Smith, L. M.Nucleic Acids Res. 1991,19, 4121-4126. Kolner, D. E.: Mead, D. .4.: Smith, L. M.Biotechniques 1992,13, 338339. Kostichka, A. J.; Marchbanks, M. L.; Brumley, R. L., Jr.: Drossman. H.; Smith, L. M. Bio/Technologv 1992,10, 78-81. (a) Heiger, D. N.; Cohen, A. S.:Karger, B. L. J. Chromatogr. 1990,516, 33-48. 0) Cohen, A. S.: Najarian, D. R; Karger, B. L. J. Chromatogr. 1990, 516, 49-60.
Ganzler, IC: Greve, K. S.: Cohen, A. S.; Karger, B. L.: Guttman, A: Cooke, N. C. Anal. Chem. 1992,64, 2665-2671. Crehan, W. A M.; Rasmussen, H. T.: Korthrop, D. M. J. Liq. Chromatogr. 1992,15, 1063-1080. Zhu. M.: Hansen. D. L.; Burd, S.;Gannon, F. J. Chromatogr. 1989,480, 311-319. Nathakarnkitkool, S.: Oefner, P. J.; Bartsch, G.: Chin, M. A,: Bonn, C. K Electrophoresis 1992,13. 18-31. Ulfelder. K. J.: Schwartz, H. E.; Hall, J. M.: Sunzeri, F. Anal. Biochem. 1992, 200, 260-267. Grossman. P. D.; Soane, D. S. J. Chromatogr. 1991,559, 257-266. Zewert, T.: Harrington, M. Electrophoresis 1993,13, 817-824. Kleemiss, M.H.; Gilges. M.: Schomburg, G. Electrophoresis 1993,14, 515522. Chrambach, A,; Aldroubi, A. Electrophoresis 1993,14, 18-22. Ruiz-Martinez. M. C.: Berka, J.; Belenkii. A: Foret, F.: Miller, A W.; Karger, B. L. Anal. Chem. 1993,65, 2851-2858. Carson, S.: Cohen, A. S.; Belenkii, A: Ruiz-Martinez, M. C.: Berka, J.: Karger. B.L. Anal. Chem. 1993,65, 3219-3226. Chrambach, A.: Rodbard. D. Science 1971,172. 440-450. 1914
Analytical Chemisfry, Vol. 67, No. 13, July 1, 1995
solution (700 000-1 000 000 M,,) and a solid (8 000 000 MA PA product is available. A separate problem is the internal coating of the capillary tubes. Without exception, the fused-silica capillaries used in DNA sequencing by CE have all been pretreated with a bonded coating. These are mostly variations of a bonded polyacrylamide layer.2g The reason is that electroosmotic flow (EOE) exists in bare fusedsilica capillaries. In the worst case, it can expel the sieving matrix from the capillary. Even when EOF is slow, the fact that it is opposite to the migration direction of DNA fragments means long separation times. Since the net motion is dictated by (LQNA p ~ o ~ the ) , corresponding difference in mobilities, the large fragments are affected much more severely than the small fragments. One therefore quickly loses the ability to read to large base numbers in the run. Unfortunately, the coating degrades with usage.2b This is not surprising since PA, when used as the sieving medium, also breaks down with time on interaction with the typical buffers used for DNA s e q ~ e n c i n g . l l ,There ~ ~ is definitely a need for better surface treatment procedures for the capillary columns to retain their integrity over many runs. In this article, we report the performance of the separation of DNA fragments from the Sanger reaction in a mixed poly(ethy1ene oxide) polymer solution in capillary columns that do not have a bonded coating.L42y EXPERIMENTALSECTION
Laser-&cited Fluorescence Detection. The experimental setup is similar to that described before.31 A high-voltage power supply (Glassman High Voltage, Whitehorse Station, NJ) was used to drive the electrophoresis. The entire electrophoresis and detection system was enclosed in a sheet-metal box with a HV interlock. A 1-mWHe-Ne laser with 543.6nmoutput from Melles Griot (Irvine, CA) was used for excitation. Two RG610 filters were used to block scattered light. The fluorescence signal was transferred directly through a l@kQ resistor to a 24bit A/D interface at 4 Hz Uustice Innovation, Palo Alto, CA; Model DT2802) and stored in a computer (IBM, Boca Raton, FL; Model PC/AT 286). Capillary and Reagents. Capillaries with 75-pm-i.d. and 365pm 0.d. were obtained from Polymicro Technologies (Phoenix, AZ). Coated capillaries were prepared by Hjerten’s method.29All chemicals for preparing buffer solutions and for coating capillaries were purchased from ICN Biochemicals (Trvine, CA); acrylamide and formamide were from Sigma Chemical (St. Louis, MO); and poly(ethy1ene oxide) was obtained from Aldrich Chemical (Milwaukee, WI). Fuming hydrochloric acid was obtained from Fisher (Fairlawn, NJ). Polyacrylamide solution (10% v/v in water, 700 000-1 000 000 M,,) was obtained from Polysciences (Warrington, PA). PGEM/U DNA samples were obtained from Nucleic Acid Facilities (Iowa State University, Ames, IA). Gel and B&er Preparations. The buffer solution was prepared by dissolving 89 mM tris(hydroxymethy1)aminomethane 0 , 8 9 mM boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA), and 3.5 M urea in deionized water. The sieving matrix was prepared by gradually adding 1.5g of 8 000 000 M,, poly(ethy1ene oxide) (PEO) and 1.4 g of 600 000 M,, PEO in 100 mL of buffer solution at 50-60 “C. During the (29) Hjerten. S.J , Chromatogr. 1985,347, 191-198. (30) Starita-Geribaldi, M.;Houri, A: Sudaka. P. Electrophoresis 1993,14, 773781. (31) Chang, H.-T.:Yeung, E. S.J. Chromatogr., in press.
addition of PEO, a magnetic stirring rod was used at a high setting to enhance the dissolution of the polymer powder. After the addition was complete, the solution was stirred for another 30 min. Then the solution was degassed in an ultrasonic bath for 30 min. Capillary Wall Treatment. The bare capillary, typically 4 5 cm total length (35cm effective length) was flushed with methanol for 10 min and then 0.1 N HC1 for 2 h, filled with a low-viscosity polymer solution (e.g., 0.5%PEO), and then filled with the polymer matrix with a syringe. The capillary was then equilibrated at the running voltage (12 kV) for 10 min before sample injection. The DNA sample was denatured by heating in a denaturing solution (5:l formamide-50 mM aqueous EDTA solution) at 95 "C for 2.5 min, and the injection was performed at 6 kV for 12 s. Between runs, the used polymer matrix was flushed out of the capillary with high pressure (400 psi, 3 min), rinsed with 0.1 N HCl for 15-30 min, and then filled with new polymer matrix. Base Calling. The base calling was performed according to the method of Li and Y e ~ n g .Briefly, ~~ this involves using the ratio of emission intensities recorded through two different optical filters. Independent confirmation was accomplished by comparison with data obtained on a commercial DNA sequencer ( B O . RESULTS AND DISCUSSION Novel Sieving Medium. In the earlier stages of our work, we used protocols that are in the l i t e r a t ~ r e to ~ ~prepare , ~ ~ noncross-linked PA solutions for DNA separations. Due to the inexperience of our personnel and the lack of direct training, e.g., in one of the leading CGE laboratories, we were not able to consistently produce sieving matrices suitable for DNA sequencing. We therefore explored the use of alternative matrices that may serve the same function. To form a sieving medium, the concentration of polymers has to be higher than a certain value called the overlap thresholdF2 Polymer chains then interact with one another to form an entangled solution. In order to create a small mesh, one wants to use a polymer with short chains and vice versa. Poly(ethy1ene oxide) with a large range of molecular weights from 300 OOO to 8 OOO OOO is available commercially. In addition, it is easy to prepare homogeneous solutions of these in typical electrophoretic buffers to provide highly reproducible separation matrices.31 Initially, to evaluate the applicability of this alternative polymer matrix for DNA sequencing, we studied the separation performance by using the ds fragments of pBR 322 DNWHueIII digest (intercalated with ethidium bromide and detected by fluorescence excited at 543 nm).31 This sample provides a good range of fragment sizes in a simple electropherogram to allow quick comparisons. It should be noted that DNA fragments from standard Sanger reactions are denatured single-stranded and covalently tagged rather than double-stranded and intercalated with a fluorophore. One actually expects ss-DNA separations to be even more efficient because there will not be a distribution of conformations or fluorophore numbers per DNA fragment. We found that the smaller fragments can be well separated in matrices prepared from individual polymers with M,, of 600 OOO2 OOO OOO. For DNA fragments from 80 to 400 base pairs, better resolution is achieved in matrices prepared from polymers with higher M,. No single polymer size provided adequate efficiencies over the entire size range. Drawing from gradient elution in (32)
Li, Q.;Yeung, E. S., unpublished results.
chromatography, we next tested mixtures of polymer sizes for the same ~ e p a r a t i o n .In ~ ~the mixed-polymer matrices, a polymer network with random pore sizes is formed. This provides optimum pore sizes for a large range of DNA fragments. The mixed-polymer matrix made from equal amounts of polymers (0.6%) with different molecular masses provided comparable resolution, while retaining a much lower viscosity, compared to all other matrices (made from single polymers) used in that study?' Further optimization of the ratio between the amounts of low-M,, and high-M,, materials in the mixed solutions should lead to even better separation performance. We have successfully used these mixed polymers for actual DNA sequencing by CE. Here, we will provide a direct comparison between PEO and 6%T non-cross-linked PA. The test sample was the set of DNA (PGEM/U) fragment ladder prepared by the Iowa State University Nucleic Acid Facility using cycle sequencing and the standard dye-labeled terminators (AB1 DyeDeoxy) and Taq polymerase. The sample preparation procedure was not altered in any way from that used to produce samples for the commercial DNA sequencing instrument (AJ3I). The injected sample is identical in concentration and composition to those suggested for loading into the commercial instrument. The test sample was independently analyzed (sequenced on the commercial instrument) and was found to be well-behaved. We have developed a matrix based on 1.4% PEO 600OOO Mn,1.5%PEO 8 OOO OOO M,,1 x TBE (PH 8.2), and 3.5 M urea for DNAsequencing. Apparently, the intermediate M,,polymers31 are not needed because the polymers at the two extremes of the size range can entangle in such a way to form the intermediate pore sizes as well. This binary matrix provides a performance very similar to the 0.7%multiple polymer matrix3I but has even lower viscosity (1200 CP at room temperature) than the 0.7%mixture. While further optimization should be possible, all experiments below were performed in this particular matrix. For comparison, commercial noncross-linked PA (10% T solution, 700 OOO-1 000 OOO MJ was diluted to form a 6%T, 1 x TBE, 3.5 M urea matrix. This solution has a measured viscosity of 4900 CP at room temperature. This is in the general range of viscosities for home-prepared material,26although it is difficult to pin down the average molecular weights for each preparation. Otherwise identical conditions were used throughout. Excitation by a He-Ne laser at 543 nm and two RG610 long-pass filters select primarily the C and T fragments. The results are shown in Figures 1 and 2 for the regions of 24-108 and 2420 bp, respectively. Presently, the resolution is insuflicient for identifying the bases beyond 420 bp. In constructing Figures 1 and 2, the ordinate of each electropherogram was adjusted to roughly match each other and to emphasize the small peaks, as those would cause the most problems in base calling due to overlap and inadequate S/N. All peaks were in fact on scale; they were merely truncated in the figures to allow plotting one on top of another. The abscissa of each electropherogram has also been adjusted to plot the same base pair region in each case. However, the actual time scale for each is different and is listed in the captions. F i r e lA,B shows that, for the small fragments, PEO provides a resolving power quite close to that of PA. PA is able to resolve certain partially overlapped peaks as well as shoulders next to large peaks in the first half of the plots. It should be noted that the run in Figure lA does provide adequate resolution for base ~alling.3~ The major difference is separation speed. The PEO Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
1915
Figure I. Separation of DNA fragments from Sanger reaction by CE from base 26 (leftmost peak) to base 108 (rightmost peak). The span of the abscissa is different in each case and is specified below: (A) PEO in coated capillary, 14-19 min; (B) PA in coated capillary, 37-55 min; (C) PEO in fresh bare capillary, 15-20.3 min; (D) PEO in HCI-reconditioned bare capillary, sixth run, 9.5-13 min; (E) PA in bare capillary, second run, 14.5-26.5 min.
plot (A) was from 14 to 19 min while the PA plot (l3) was from 37 to 55 min. This is expected from the higher viscosity of the PA matrix. Very striking is the difference in separation for the large fragments, Figure 2A,B. The PEO matrix clearly provides better resolution and may even be extending the convergence limit to higher base numbers. This is important because the further one 1916 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
can call bases in a run the less front- and back-end work will be needed. Figure 2 4 B again shows that separations run much faster in PEO than in PA. In the middle range (108-420 bp, data not shown), there is a one-to-one correspondence between the resolution of DNA fragments in PEO compared to that in PA, although some degradation is already evident in PA past 320 bp.
- -
Time Figure 2. Separation of DNA fragments from Sanger reaction by CE from base 420 (leftmost peak) upwards. The span of the abscissa is different in each case and is specified: (A) PEO in coated capillary, 39-52 min; (B) PA in coated capillary, 111-129 min; (C) PEO in fresh bare capillary, 40-52 min; (D) PEO in HCI-reconditioned bare capillary, sixth run, 26-33 min; (E) PA in bare capillary, second run, 60-69.5 min.
It should be noted that our migration times for PA (Figures 1B and 2B) cannot be directly compared with the work of others because of differences in the way the PA matrix and the column coating was prepared. Comparisons among the times for the individual traces in Figures 1 and 2 are however valid. There is a reasonable explanation for the differences in performance. Apparently, the maximum length of our PA polymer
(up to 1OOO OOO MA is not sufkient to form dynamic pore sizes large enough for the large DNA fragments. In fact, a PEO molecule of the same M, should be longer than PA because of the specific atomic arrangement along the backbone. The same is true when PEO is compared with any other polymer that has been used for CE sieving. A related polymeric material is poly(ethylene glycol) (PEG). Structurally, PEG is almost identical to Analytical Chemistty, Vol. 67, No. 13, July 7, 1995
1917
PEO; only the starting monomer and the polymerization process are different. The latter is probably the main reason why commercial PEG preparations are not available out to the millions of daltons that PEO can be purchased at. Incidentally, any of the PEO polymers costs only around $1O/g, and we are using 2-3% solutions in the 2-pL range per capillary. Other workers26,z7,33 have demonstrated base calling in CE with 6% T PA out to '400 bp. Presumably, the home-prepared PA matrices have higher Mn components and thus include larger pore sizes in solution. The low catalyst concentration and long polymerization times at low temperature9 support this argument. In fact, it may well be true that a wide range of polymer lengths are (fortuitously) produced to allow non-cross-linked PA to separate a large range of DNA sizes with good efficiency. We tried to mix our PA solution with some 8 000 000 MnPA from the same manufacturer. However, we were not able to obtain a homogeneous mixture for testing its performance. Column Treatment Protocol. The problems with the protective coating on the internal wall of the capillary have been discussed above. Even though in principle one can replace the sieving medium after every run to allow repeated usage of the , * ~are , ~ not ~ capillaries, the coatingz9 gradually d e g r a d e ~ . ~ ~We aware of any report where a capillary column has been used for tens of runs in DNA sequencing. One thought is to regenerate the coatingz9after several runs by repolymerization of PA in situ. Our experience is that the original performance cannot be restored in this manner. Another thought is to avoid the PA coating, which definitely will degrade in We attempted to cap the silanol groups on the fused-silica wall by treating with [3-(methacryloxy)propyl]trimethoxysilane (silanization) and use the capillaries without further polymerization with PA. The electroosmotic flow was indeed substantially reduced, as judged by the migration times of the primer peak and the high molecular weight convergence peak. However, the separation efficiency was compromised to the extent that calling bases beyond 200 bp was not possible. Since the main purpose of coating the capillary column is to eliminate electroosmotic flow, we proceeded to evaluate alternative approaches toward this goal. In the zone electrophoretic separation of small molecules and even proteins, it is common to use a dynamic coating of a linear polymer such as PEG, hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, or PEO. The electrophoretic buffer contains a dilute solution of these to continuously replenish the adsorbed polymer at the wall. This suggests that DNA sequencing can be performed on a bare fusedsilica column. The sieving polymer will also dynamically coat and isolate the capillary walls. Even if there is some residual surface charge, the high viscosity of the polymer matrix will lead to further reduction of this undesirable counterflow during DNA separation3 We therefore started with commercial capillary columns and washed out the inside with methanol. Immediately afterward, the polymer matrix was introduced into the capillaries and a DNA sequencing run was started. DNA separations in bare capillary columns are shown in Figures 1C and 2C for the PEO mixed-polymer matrix. There is practically no difference in the electropherograms with or without a coating on the capillary wall (comparing plots C with plots A). Even the actual migration times are almost identical. This is the (33) Best, N.; Arriaga, E.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1994,66, 4063 -4067. (34) Lee, T. T.: Yeung, E. S. Anal. Chem. 1991,63,2842-2848.
1918 Analytical Chemistry, Vol. 67,No. 73,July 7, 7995
first demonstration of DNA sequencing in CE without a bonded coating on the column wall. Unfortunately, while we can reproduce these results every time for a new capillary column, the resolution invariably started to degrade immediately (after one or two runs). The migration times became progressively longer and the largest fragment peaks became unrecognizable. Naturally, having to replace the capillary array35after every run is not a viable option for high-throughput DNA sequencing. With the surprising results in Figures 1C and 2C, we attempted to regenerate the original column surface after a run by flushing with deionized water, methanol, buffer solution, 1 M NaOH, and combinations of these. In no case was the original performance restored. Finally, we realized that the symptoms associated with column degradation are those of increased electroosmotic flow in subsequent runs. For a dry,fresh column (fused-silica) surface, the silanol groups should be fully protonated. A methanol wash further minimizes any adsorbed moisture. So, initially the electroosmotic flow should be negligible. The viscous polymer matrix has the advantage of reducing the effects of any residual surface 5 potential34on bulk flow. However, since the separation matrix is at pH 8.2, it will eventually cause ionization of the silanol groups to increase the electroosmotic flow. The remedy is to flush the column in between runs with 0.1 N HCl, to retitrate the surface silanol groups back to their original protonated state. The performance of a bare fused-silica CE column in separating DNA fragments after six cycles of PEO fill, DNA electrophoresis, pressure removal of PEO, and 0.1 N HCl reconditioning is shown in Figures 1D and 2D. There is no obvious difference in resolution between this electropherogram and those for coated column/PEO (Figures lA and 2A) or for a fresh bare column/PEO (Figures 1C and 2C), except for a somewhat earlier onset of the convergence limit. This is in a region of '600 bp, where the resolution is not sufficient for calling bases and where the Taq polymerase reaction is not expected to be reliable anyway. These results are very recent; so far we have observed reproducible performance in separation efficiency over 30 runs in a 3-week period. There are slight random variations in migration times (12%for different batches of matrix, different columns, different days), but no systematic change over time. This is to be expected since we relied on a manual flush-fill operation and have not yet defined the time and concentrations required to completely rejuvenate the column surface. Since no bonded coating is used, there is nothing to degrade and the column should in principle last indefinitely. This is the first demonstration of extended usage of a CE capillary for DNA sequencing. A surprising result is that the migration times observed in the HC1-treated capillaries are much shorter than those found for any polymer matrix-surface preparation, either reported here or in the literature. One possible explanation is that the coating (bonded) on the capillaries and the fresh fused-silica surface both have residual electroosmotic flow to oppose DNA migration. The HCl treatment was able to eliminate electroosmotic flow entirely. Regardless of the mechanism, the bottom line is that bases 28420 eluted within a time span of only 16 min for an average rate of 25 bp/min. This is faster by a factor of 3-5 compared to reported results using non-cross-linked PA in coated capillaries. Finally, it is interesting to see how a noncross-linked PA matrix performs in a bare fused-silica capillary. This is shown in Figures 1E and 2E. Resolution for the small fragments (Figure 1E) is (35) Ueno, K.: Yeung. E. S. Anal. Chem. 1994,66, 1424-1431.
the best of all the systems studied here, but the resolution beyond 420 bp (Figure 2E) is the worst. This electropherogram is actually the second run on the bare column, indicating that degradation is slower than for the case of the PEO matrix on a bare column. There was still gradual degradation due to an increase in electroosmotic flow, as the first run started 0.5 min earlier and ended 10 min earlier. This is consistent with the fact that PA is a more viscous matrix, so it takes longer for the ions in the bulk medium to titrate the surface silanol groups to the same extent. Unfortunately, the same HC1-reconditioning procedure was not effective after a column was filled with PA. The implication is that we were not able to comptetely flush out this more viscous matrix to allow HCl to react with the surface effectively. Another possibility is that HCl causes rapid decomposition of PA1lz3Oand contaminates the column surface. System Integration. In anticipation of the need to flush out, recondition, and refill the capillary columns after each of many runs and the need to inject multiple samples separately into the array,35 we have developed a pressure cell suitable for these operations. Note the pressure needed for this relatively lowviscosity sieving matrix is only 100-400 psi, depending on the time allowed for each operation. The cell shown in Figure 3 has been used for singlecapillary operation in most of the experiments described above. For 100 capillaries in a bundle, the exit end can be gathered together to form a close-packed group of only 2 mm in diameter. This can go through the same fitting as shown in Figure 3 to implement the same recycling process. It is worth noting that we have obtained results virtually identical to those in Figures lA and 2A in runs derived from pressure injection of the DNA sample in the pressure cell (100 psi, 20 s). The high viscosity of the polymer matrix means that only a very narrow plug of sample was injected in a controlled manner. The electropherograms showed the same resolution as the electrokinetically injected runs in the traces in Figures 1 and 2. Actually one would expect that pressure injection will avoid (36) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992,64, 1226-1231
O-ring
nut
bolt
m
inlet
1
1
+/#
p l e x i g k block
Figure 3. Pressure injection-flush cell for one capillary. About 100500 psi can be used. Fittings are standard liquid chromatographic plumbing.
the electroinjection bias36that discriminates against the larger DNA fragments. Evaluation of such an advantage will be performed in the next phase of this project. Finally, pressure injection avoids cross contamination of the sample from the electrodes and avoids the need to make electrical contacts with each sample vial in a large array entirely. ACKNOWLEDGMENT The Ames Laboratory is operated for the U S . Department of Energy by Iowa State University under Contract W-7405-Eng-82. This work was supported by the Director of Energy Research, Office of Health and Environmental Research. Received for review March 1, 1995. Accepted April 15,
1995.B AC950217P a Abstract published in Advance ACS Abstracts, May 15, 1995.
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