Anal. Chem. 1992, 64, 1221-1225
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Preparation of Polyacrylamide Gel Filled Capillaries for Ultrahigh Resolution of Polynucleotides by Capillary Gel Electrophoresis Yoshinobu Baba,' Toshiko Matsuura, Kyoko Wakamoto, Yoshiko Morita, Yumi Nishitsu, and Mitsutomo Tsuhako Kobe Women's College of Pharmacy, Kitamachi, Motoyama, Higashinada-ku, Kobe 658, Japan
A method for the productlon of polyacrylamlde gel fllled caplllarles was studled In detail. A polymerlzlng solution of acrylamlde was Injected Into the caplllary wlthout Its Inner surface pretreatmat and polymerlzed, In sltu, by radical Inltlators. Bubble fonnatlonIn caplllarles was avdded by uslng welldeslgned lnjectlon equlpment, which was developed for use In thls study. Performance of gel-filled caplllarles was examlnad In terms of dablllty, reproduclbllltyof mlgratlontlme, feadblllty of method, and resolving power of polynucleotides. Gel-fllled caplllarles prepared by thls method showed hlgh preclslonIn relatlve mlgratlontime, ultrahlghresotutlon of polynuCMMe8, and wlde applkablltly. Average relatlve standard devlatlons In migratlon tlmes for polynucleotldesIn the chaln length range from 50 to 250" were 1.1% (run to run), 1.5% (day to dey), and 2.1 % (batch to batch), respectlvely. Stablllty of the gel-fllled caplllary was less than that of a gelfllled caplllary In whlch the gel was chemlcally bound to the capllary Inner surface. A plate number for a gel-fllled caplllary of 1.5 X lo7 m-l was achleved. Mlxtures of 450 klnds of pdyadenylk adds were basellnwesolvedand analyzed wlthln 100mir,, Hlgh-resolutlonseparationof a mlxtureof pdydeoxyadenyllc aclds was also achleved. The method was demonttrated to be appllcable to the productlon of gel-fllkd caplllarleswtlh wlde varletles of gel composltlonand caplllary dlameters. Advantages and llmltatlons of thls method are dlrcuased.
INTRODUCTION Cohen et al. and Guttman et a1.192 first demonstrated highspeed single-base resolution of DNA polynucleotides by capillary gel electrophoresis (CGE) utilizing a polyacrylamide gel filled capillary, i.e., capillary polyacrylamidegel electrophoresis (C-PAGE). The resolving power and speed of C-PAGE have been illustrated to be much better than those of slab gel electrophoresis3* and HPLC.7 DNA Sequencing technology using C-PAGE,3+ therefore, appears to be an attractive alternative to the conventional automated DNA (1)Cohen, A. S.;Najarian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A,; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988,85,9660-9663. (2)Guttman, A.; Cohen, A. S.;Heiger, D. N.; Karger, B. L. Anal. Chem. 1990,62,137-141. (3)(a) Drossman, H.; Luckey, J. A,; Kostichka, A. J.; D'Cunha, J.; Smith. L. M. Anal. Chem. 1990.62.900-903. (b) Luckev, J. A.: Drossman, H.; Kostichka, A. 3.; Mead, D. A.; D'Cunha, J.; Norrh, T. B.;Smith, L. M. Nucleic Acids Res. 1990,18,4417-4421. (c) Smith, L. M. Nature 1991,349,812-813. (4)(a) Swerdlow, H.; Gesteland, R. Nucleic Acids Res. 1990,18,14151419. (b) Swerdlow,H.; Wu, S.;Harke, H.; Dovichi, N. J. J.Chromatogr. 1990,516,61-67. (5)Cohen, A. S.;Najarian, D. R.; Karger, B. L. J.Chromatogr. 1990, 516,49-60. (6)Brennan, T.; Chakel, J.; Bente, P.; Field, M. Biological Mass Spectrometry; Elsevier; Amsterdam, 1990;pp 159-177. (7)Baba,Y.;Mat.auura,T.; Wakamoto,K.;Tsuhako,M. J. Chromatogr. 1991,558,273-284. 0003-2700/92/0364-122 1$03.00/0
sequencers based on slab gel electrophoresis because of improved speed, resolution, and DNA sequencing efficiency. Several authors report that C-PAGE can sequence over 300 bases in a 1-h run with high sensitivity at a detection limit of mol of fluorescein-labeled DNA using laser-induced fluorescence detection.3-5 Smith et al.3b first performed sequence analysis of DNA by simultaneous detection of four different fluorescently tagged primers with a single capillary run, whereas other groups have sequenced DNA with four runs which detected four bases independently.'+ A method for the production of polyacrylamide gel filled capillaries is the key technology needed to advance highspeed DNA sequencing.& Several researchers1-7-+15have been investigating the methodologies, and some commercial gelfilled capillary columns have recently become available from Applied Biosystems, Beckman Instruments, and J & W Scientific. However, a simple and reliable method for the production of bubble-free gel-filled capillaries has not yet been developed. In preliminary experiments,16we demonstrated a method for the production of bubble-free polyacrylamide gel filled capillaries, which realized high-speed DNA separation with single-base resolution of polynucleotides up to 250 bases within 60 min. In the present paper, we demonstrate that the capillaries produced by this method achieve ultrahigh speed and resolving power in the single-base resolution of polynucleotides. The method is based on injection of a polymerizing solution into uncoated capillaries and in situ polymerization. The reliability of the method is shown by the reproducibility and stability of gel-filled Capillaries. To demonstrate the general applicability of the method, we made gel-filled capillaries with differing capillary diameters and gel compcc.kions and separated a complex mixture of poly(adeny1ic acids). High-performance separations of polynucleotides demonstrated that the resolving power of the gel-filled capillaries prepared by this method was comparable to the resolving power of those prepared by other methods.l-7,"-16 Similar methods have been proposed by other re~earchers;~J3 however, this is the first report in which the advantages and the limitations of the method have been studied systematically and the feasibility and performance demonstrated. (8)Trainor, G.L. Anal. Chem. 1990,62,418-426. (9)Karger, B. L.;Cohen, A. S. Eur. Pat. Appl. EP324539,(Northeastem University) July 19, 1989; U S . Pat. 4,865,706,Sept. 12,1989;U.S. Pat. 4,865,707,Sept. 12,1989. (10)Bente, P. F.; Myerson, J. (Hewlett-Packard Co.) Eur. Pat. Appl. EP272925,June 29, 1988; US. Pat. 4,810,456,March 7, 1989. (11)Paulus, A,; Ohms, J. I. J. Chromatogr. 1990,507,113-123. (12)Heiger, D. N.; Cohen, A. S.;Karger, B. L. J. Chromatogr. 1990, 516,33-48. (13)Lux, J. A,; Yin, H.-F.; Schomburg, G. J. High Resolut. Chromatogr. 1990,13,436-437. (14)Yin, H.-F.; Lux, J. A.; Schomburg, G. J. High Resolut. Chromatogr. 1990,13,624-627. (15)Baba, Y.; Tsuhako, T.; Enomoto, S.;Chin, A. M.; Dubrow, R. S. J. High Resolut. Chromatogr., 1991,14,204-206. (16)Baba, Y.; Matsuura, T.; Wakamoto, K.; Tsuhako, M. Chem. Lett. 1991,371-374. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
EXPERIMENTAL SECTION Materials. Tris(hydroxymethy1)aminomethane (Tris),boric acid, and urea (Wako, Osaka, Japan) were of analytical grade. Acrylamide, N,"-methylenebis(acry1amide) (BIS),N,N,N',N'tetramethylethylenediamine (TEMED),and ammonium peroxydisulfate (Wako) were of electrophoretic grade. Nuclease P1 was a product of Yamasa (Chiba, Japan). Poly(adeny1ic acid) [poly(A)] was purchased from Yamasa and Pharmacia LKB (Uppsala, Sweden). Poly(deoxyadeny1ic acid) [poly(dA)] was obtained from Sigma (St. Louis, MO) and Pharmacia. Poly(deoxyadenylic acids) of chain lengths from 1 2 to l8mer and poly[p0ly(dA)~z-~8] and from 40 to 6Omer [poly(dA)-] (adenylic acids) of chain lengths from 12 to l8mer [p0ly(A)~z-~8] were supplied by Pharmacia. The concentrations of samples in aqueous solutions were 2.5 units/lOOpL for poly(dA)lz-ls,5 units/ lOOpL for poly(dA)-, and 5 unita/100pL for poly(A)12-18.These polynucleotide samples were stored at -18 "C before use. Preparation of Poly(A) and Poly(dA) Enzymatic Partial Hydrolysate. Oligoadenylate fragments from poly(A) were prepared by enzymatic hydrolysis of poly(A) with nuclease P1. Poly(A) (30 mg) was dissolved in 1mL of a citrate buffer solution (0.3 M, pH 6). An aliquot (2 pL) of an aqueous solution of nuclease Pl(1pg/mL) was added to an aliquot (50pL) of the buffered solution of poly(A), and the resultant solution was allowed to react at 40 "C for 10 min. Oligodeoxyadenylate fragments from poly(dA)were prepared in a similar manner. Poly(dA) (2.5 units) was dissolved in 10pL of a citrate buffer solution (0.3 M, pH 5.3). An aliquot (2 pL) of the aqueous solution of nuclease P1 (1pg/ mL) was added to the buffered solution of poly(dA), and the resultant solution was allowed to react at 40 "C for 2 min. The polynucleotide samples were stored at -18 "C before use. Enzymatic digestion of poly(A)or poly(dA)described above gave a mixture of polynucleotides containing a 5' terminal phosphate in the chain length range from monomer to 300mer.7J6 Poly(A) or poly(dA)enzymatic partial hydrolysate is of greater advantage than the poly(dA)- sample, which is widely used for the CPAGE as a model substrate to demonstrate the resolving power of C-PAGE, because enzymatic digestion of polynucleotides produces a mixture containing a broader chain length range of polynucleotides. CE Instrumentation. C-PAGE separations were carried out by using an Applied BiosystemsInc. (ABI,Foster City,CA) Model 270A capillary electrophoresis system. The electropherograms were processed on a Hitachi (Tokyo, Japan) D-2500 integrator. Polyimide-coated fused-silica capillaries (375-pm o.d., 50-, 75-, and 100-pm i.d.) were purchased from Polymicro Technologies (Phoenix, AZ) and GL Sciences (Tokyo, Japan). Polyacrylamide gel filled capillaries were prepared by the method described in the subsequent section. Gel-filled capillaries were mounted in the AB1Model 270A instrument and run with a buffer solution at 10-30 kV (200-500 V/cm). The running buffer solution was the mixture of 0.1 M Tris, 0.1 M boric acid, and 7 M urea (pH 8.6). The capillary temperature was kept at 30 f 0.1 "C. The polarity was reversed, ensuring injection of the samples at the cathode. A sample solution was injected into the capillary electrophoretically by applying a voltage of 5 kV for 1.-5 s. Polynucleotides were detected at the absorption maximum (260 nm) of poly(A) and poly(dA). Preparation of Polyacrylamide Gel Filled Capillaries. Acrylamide is polymerized in the capillary with "-methylenebis(acry1amide) (BIS) as a cross-linking agent. The polymerization reaction is initiated by the addition of ammonium peroxydisulfate. In addition, N,N,N',N'-tetramethylethylenediamine (TEMED) was added as a catalyst. Cross-linked polyacrylamide gel has a pore structure which can be varied by differing the amounts of monomer and a cross-linking agent.17 The total concentration of monomer and the concentration of the cross-linkingagent for the polyacrylamide gel are generally expressed respectively as % T and % C.18 Polyacrylamide gel filled capillaries were prepared as follows. A stock solution of acrylamide was prepared by dissolving the appropriate amounts of acrylamide and BIS in distilled water, (17) Fawcett, J. S.; Morris, C. J. 0. R. Sep. Sci. 1966, I, 9-26. (18)Hjerten, S. Arch. Biochem. Biophys., Suppl. 1962, 1, 147-151.
capillary silicone rubber pre-drilled septum
t
capillary SiiiCOne rubber drilled stopper --c
to vacuum
-_ polymerizing solution
/
distilled water
1
50-mL flask with
side hose connection
Flgure 1. Equlpment for the injection of the polymerlzlng solution for
acrylamMe Into capillaries.
e.g., 19 g of acrylamide and 1g of BIS in a 50-mL solution (40% T and 5% C). The stock solution was stored at 5 "C before use. The capillary (50-, 75, or 100-pm i.d.) without its inner surface pretreatment was cut into the appropriate lengths, e.g., 50 cm, and 0.5 cm of polyimide coating was burned off at a distance of ca. 20 cm from the outlet end. The char was cleaned off with methanol. The capillary was rinsed by passing distilled water into the capillary for 10 min by using a simple vacuum injection system, as shown in Figure 1. To introduce distilled water into the capillary, the flask was vacuumed at ca. 20 mmHg. The flow rate was 5-10 pL/min, when the 100-pm capillary was used. The stock solution of acrylamide was diluted with the running buffer solution. For example, 1 mL of the stock solution (40% T and 5 % C) was diluted with 7 mL of the buffer solution, giving a final acrylamide (5% T and 5 % C)solution. The diluted acrylamide solution was carefully degassed in an ultrasonic bath for 5 min. The solutions of 10% (v/v) TEMED and 10% (w/v) ammonium peroxydisulfate were prepared freshly. Polymerization was initiated by the addition of 10 pL of the ammonium peroxydisulfate solution and 10 pL of the TEMED solution into 5 mL of the diluted solution of acrylamide. The solution was brought to 0.02% (v/v) TEMED and 0.02% (w/v) ammonium peroxydisulfate (note, these concentrations are lower than normally used for polyacrylamidegeIa9. The polymerizing solution was quickly passed through the capillary for 5 min at the speed of 1-2 cm/s by using the vacuum injection system (Figure 1). Polymerization in the capillary was completed within 5 h at room temperature. Both ends of the gel-filled capillary should be trimmed for the reduction of the UV baseline noise. The baseline noise level of an electropherogram after the trimming was reduced to ca. one-third of the noise level before the trimming. The ends of capillary which has been thus cut are examined under a microscope to ascertain that the cutting operation produced the requisite flatness of the exposed polyacrylamide gel. Prerunning with the buffer solution at 100-150 V/cm for 20 min would be recommended for the reduction of baseline noise.
RESULTS AND DISCUSSION Equipment for Injection of the Polymerizing Solution into the Capillary. The methods, which were proposed by several authors,1~3a~48~9~10~13~14~16 for the production of a gel-filled capillary generally consisted of four steps: (1)pretreatment of the capillary inner surface; (2) preparation of a polymerizing solution of acrylamide; (3) injection of the polymerizing (19) Sealey, P. G.;Southern, E. M. In Gel Electrophoresis ofNucleic Acids, 2nd ed.; Rickwood, D.; Hames, B. D., Eds.; IRL Press: Oxford, U.K., 1990; Chapter 2.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
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Table I. Reproducibility of Migration Time of Poly(A) in C-PAGE' run-to-runb 3-1 35 rnin
yo 3i)
30
40
50
60
300
~
70
miri
Figure 2. C-PAGE separation of poly(A) digested by nuclease P1. Conditions: Capillary 100-pm i.d., 375-pm o.d., 50-cm length, 30-cm effective length; running buffer 0.1 M Tris-borate and 7 M urea, pH 8.6;gel contained 5% T and 5% C; field, 200 V/cm; current, 10 pA; injection, 5 kV for 1 s; detection, 260 nm.
solution into the capillary; (4) polymerization of the acrylamide in the capillary. Two main problems of the method remained: (1)the bubble formation during the polymerization process; (2) the stabilization of the gel in the capillary. Some researchers1~gJlemphasized that the pretreatment of the capillary inner surface was needed to stabilize the gel in the capillary. Others pointed out that the bubble formation was avoided when acrylamidewas polymerized under the mild conditions of radical initiators4aJ4J6 or the polymerizing solution was compressed and the high pressure was maintained during polymerization.3aJO In our opinion, the bubble formation could be avoided by using well-designed injection equipment for the introduction of the polymerizing solution into capillary in addition to the polymerization under the moderate conditions. Figure 1shows equipment for the introduction of the polymerization solution into the capillary. The capillary is threaded through a silicone rubber predrilled septum purchased from ABI, of which the diameter is 6 mm. One end of the capillary is passed through the hole of a drilled silicone rubber stopper and the septum is inserted into the 6-mm hole of the drilled stopper. The stopper with the capillary is mounted on the 50-mL flask containing distilled water. The capillaryis slipped up or down in the septum as necessary to position the end of the capillary no more than 5 mm from the bottom of the flask. The other end of the capillary is immersed in the polymerizing solution of acrylamide. The vacuum injection of the acrylamide solution into the capillary is advantageous for the production of bubble-free gel-filled capillaries. The bubble formation during polymerization occurs less than about 5% of the time. Although we tried to make a gel-filled capillary by the siphoning introduction of the acrylamide solution according to the literature? gas bubbles tended to form during polymerization at the rate of over 20%. Performance, Reproducibility, and Stability of the Gel-FilledCapillary. In order to examine the performance, reproducibility, and stability of the polyacrylamide gel filled capillaries prepared by the present method, poly(A) mixtures were separated under the separation conditions, as shown in Figure 2. Figure 2 clearly demonstrated that ultrahigh resolution of the poly(A) mixture was achieved by using the gel-filled capillary prepared by this method. A total of 300 bands of poly(A) were baseline-resolved within only 72 min. A time-expanded electropherogram in the migration time range from 34 to 35 min, as shown in Figure 2, illustrated that the speed of separation of five peaks per minute was realized. To determine the chain length of poly(A) for each band, p0ly(A)~2-~8 was coinjected with the poly(A) mixture. Consequently, the peaks with a migration time of ca. 20 min are assigned to poly(A) of chain lengths from 12 to 18mer. The large peak at 17 min would correspond to unseparated oligoadenylicacids of chain lengths from the monomer to 5mer. If a single-baseresolution was assumed to be achieved in all chain length ranges, the poly(A) series of chain lengths from
day-to-dayb
batch-to-batch*
chain migration RSD migration RSD migration l e n a h time(min) (%) time(min) (%) time(min) 50 51 100 101 150 151 200 201 250 251
25.16 25.36 34.63 34.83 44.50 44.70 54.15 54.33 63.20 63.39
0.83 0.82 0.93 0.92 1.2 1.2 1.1 1.1 1.6 1.5
25.40 25.59 34.63 34.83 44.24 44.43 53.56 53.74 62.07 62.24
1.4 1.3 0.66 0.66 1.2 1.2 1.7 1.7 2.3 2.4
25.60 25.81 35.00 35.19 44.71 44.91 53.67 53.99 62.16 62.33
RSD
(74) 1.8 1.9 2.2 2.2 2.7 2.7 1.6 1.7 2.0 2.1
Conditions: capillary 100-pm i.d., 375-pm o.d., 50-cm length, 30-cm effective length; running buffer 0.1 M Tris,O.l M boric acid, and 7 M urea, pH 8.6; gel contained 5% T and 5 % C; field, 200 V/cm; current, 10 pA. b n = 5.
6 to 305mer could be completely resolved. The chain length of such a large poly(A), however, could not be determined, because of the lack of authentic sample and the difficulty in the preparation of large RNA samples. The plate number of each peak was estimated to be 2.3 X lo6 (7 X 106 m-1) for peak number 30,1.5 X 106 (5 X lo6m-l) for peak number 50, and 9.6 X 105 (3 X 106 m-1) for peak number 100, respectively. This high efficiency, which is obtained by using C-PAGE without optimization of the capillary size, the gel composition, and the applied field compares favorably with the reported plate numbers of (330) X 106m-l,1~2~3b~4a,7,16 which are achieved under the optimum conditions. The reproducibility of migration time was examined by using of the gel (5 % T and 5 % C) filled capillary of Figure 2. The migration time of poly(A) in the chain length range from 50 to 251mer was measured several times a day. The measurements were repeated everyday for severaldays. Table I lists the resultant values of the relative standard deviation (RSD). The RSD values for the run-to-run reproducibility of the migration time ranged from 0.82 to 1.6 % RSD (n = 5). The average value in the chain length range from the 50 to 251mer was 1.1% RSD. The reproducibility of relatively low molecular weight poly(A)was slightly better than that of high molecular weight poly(A). The average values of the dayto-day and the batch-to-batch reproducibility were 1.5 % RSD ( n = 5) and 2.1% RSD ( n = 5), respectively. These RSD values were comparable to those reported by using gel-filled capillary in which the gel was chemicallybound to the capillary inner surface.2 Each band spacing and the plate number were unchanged so that the high resolving power of the gelfilled capillary as illustrated in Figure 2 was maintained during the measurements. These data demonstrate that the present method is suitable for the production of a reproducible gelfilled capillary which shows ultrahigh resolving power for polynucleotides. We next examine the stability of the gel-filled capillary. The stability was affected strongly by the sample matrix, the applied field, and the gel concentration. Gel-filled capillaries gave only a several percent decrease in the plate number after 50 injections of the p01y(dA)~msample using a polyacrylamide gel (5% T and 5% C) filled capillary by applying an electric field of 200 V/cm. However, a significant loss in performance was observed after 10-20 injections of a poly(A) mixture, of which the separation is shown in Figure 2. In addition, an increase in the applied field led to a decrease in the stability; e.g., the separation performance decreased after 5-10 injections of the poly(A) mixture at 300 V/cm or 1-5 injections at 400 V/cm, using the same gel-filled capillary.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
A 100 pm
10
20
40
30
50
70 min
60
Figure 3. C-PAGE separations of poly(A) digested by nuclease P1. Separation conditions are as in Figure 2 except for capillary diameter. Current: 3 (50 pm), 7 (75 pm), and 12 pA (100 pm).
40
50
/U
8U
70
~~
I
3% T and 5% C
Iuu
YU
Time (min)
Flgure 5. C-PAGE separation of poly(A) digested by nuclease P1. Conditions: capillary 100-pm i.d., 375-pm o.d., 50-cm length, 30-cm effective length; running buffer 0.1 M Tris-borate and 7 M urea, pH 8.6; gel contained 5% T and 1.5% C; field, 200 V/cm; current, 12 pA; injection, 5 kV for 1 s; detection, 260 nm.
50
18
20
22
24
26
28
30
32
34 min
Flgure 4. Effect of polyacrylamide gel concentration on C-PAGE separations of poIy(dA)40-80.Separation conditions are as in Figure 2 except for gel composition.
The stability was also influenced by gel concentration (% T). Lifetime of the 3% T gel-filled capillary was reduced to ca. half of that of the 5% T gel-filled capillary. The electroosmotic flow arising on the untreated inner surface of the capillary can cause the gel to slowly migrate out of the capillary48J4 Since the 3% T gel would migrate readily compared with the 5% T gel, the stability of the lower percentage gel-filled capillary decreased. The separation performance could be restored by cutting off a few millimeters of the capillary.11J4 The stability of the gel-filled capillary prepared by the present method was less than that of the gel-filled capillary in which the gel was chemically bound to the capillary inner surface by using a bifunctional reagent such as (3-(methacryloxy)propyl)trimethoxysilane,1~~J1J4 the capillary which could be used for 150-200 measurements. The results, however, showed that relative stable gel-filled capillariescould be obtained by this method without capillary inner surface pretreatment. To demonstrate the wide applicability of the method, we made gel filled capillaries with differing capillary diameters and gel compositions. The equipment in Figure 1was easily applied to the production of the gel-filled capillary of 50- or 75-pm diameter. Figure 3 shows the separation of the poly(A) mixture at 200 V/cm, using the gel (5% T and 5% C) filled capillary of 50-,7 5 , or 100-pmdiameter. The resolving power of the gel-filled capillary of 50- or 75-pm diameter was as high as that of the gel-filled capillary of 100-pm diameter. We further examined the applicability of the method to the production of the gel-filled capillaries with differing gel compositions. Gel-filled capillaries in the gel composition range from 1.5 to 5% C and from 2 to 10% T were easily prepared by the present method. Figure 4 shows the example of the separation of the poly(dA)4Mo sample with gel concentrations differing from 3 to 7% T, holding the % C constant. The result demonstrates that the separation speed and the resolution strongly depend on the gel density. The method presented here was elucidated to produce the reproducible gel-filledcapillarieswhich showed high resolving power and to be applicable to the production of gel-filled
I
10
15
100
20 150
200
I
20
25 250
30 300
Time (min)
Figure 6. C-PAGE separation of poly(dA) digested by nuclease P1. Conditions: capillary 75-pm i.d., 375-pm o.d., 50-cm length, 30-cm effective length; running buffer 0.1 M Tris-borate and 7 M urea, pH 8.6; gel contained 3 % T and 3% C; field, 200 V/cm; current, 11 pA; injection, 5 kV for 1 s; detection, 260 nm.
capillaries with wide varieties of capillary diameters and gel compositions. We next turn to the use of the gel-filled capillaries for the high-speed separation of a polynucleotide mixture. High-ResolutionSeparation. Ultrahigh performance of the gel-filled capillary is demonstrated by the separation of a broader chain length range of poly(A) (Figure 5 ) and the high-speed separation of a poly(dA) mixture (Figure 6), in order to examine whether the present method can be applied to the routine DNA sequencing. A total of 450 bands of poly(A) were completely resolved within 100min a t 200 V/cm, as shown in Figure 5, using the gel ( 5 % T and 1.5% C) filled capillary of an effectivelength of 30 cm. This poly(A)mixture was prepared by the method described in the Experimental Section except for a half-reduction of the concentration of nuclease P1. The plate number of 300mer in Figure 5 was calculated to be 1.5 X lo7 platedm and that of 150mer was 1 X 107 plates/m. We next turned to design the capillary filled with the lower percentage T gel for higher throughput. High-speed separation (Figure 6) was achieved by using the gel (3% T and 3% C) filled capillary, showing the complete separation of 300mer of poly(dA) from 30lmer within only 38 min. The plate number of 250mer was calculated to be
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992 Table 11. C-PAGE'
Migration Time of Poly(A) and Poly(dA) in
chain length
poly$4) migration time (min)
poly(dA) migration time (min)
50
18.50 18.57 22.03 22.10 25.69 25.76 29.48 29.55 33.34 33.41 37.21 37.28
18.46 18.52 21.96 22.04 25.76 25.84 29.63 29.70 33.60 33.68 37.65 37.72
51 100 101 150 151 200 201 250 251 300 301
1225
a Conditions: capillary 100-pm i.d., 375-pm o.d., 50-cm length, 30-cm effective length; running buffer 0.1 M Tris, 0.1 M boric acid, and 7 M urea, pH 8.6; gel contained 3 7% T and 3 5% C; field, 200 V/cm; current, 10 pA.
1.5 X lo7plates/m. A poly(A) mixture was also separated by using the same capillary under the same separation conditions and the migration time of poly(A), i.e., a RNA homopolymer, was compared with that of poly(dA), Le., a DNA homopolymer, as listed in Table 11. The migration time of a DNA homopolymer was almost comparable to that of a RNA homopolymer in the chain length range from 50 to 30lmer. This shows that a DNA homopolymer can be separated under the same conditions for the separation of a RNA homopolymer. However, some different strategies may be required for the separation of a DNA heteropolymer, such as DNA sequence reaction products, of which the separation is sometimes plagued by compressions.3b The plate number and the separation speed realized in Figures 5 and 6 are roughly comparable to the reported plate numbers (3 X 107 by Guttmanetal.2and1.3 X 107byLuckeyetal.3b)andthethroughput in the DNA sequence analysis by using C-PAGE.34 These results illustrated that gel-filled capillaries produced by the present method would be applicable to the high-speed DNA sequencing. The migration time of poly(A) measured from Figure 5 was plotted versus the base number of poly(A), as shown by the open circles in Figure 7,to test the linearity of the plot in the chain length range from monomer to 450mer. The plot results in a straight line with a slope of 6.4 and a correlation coefficient of 0.999. Other plots obtained by using gel-filled capillaries with differing 9% T gave the straight lines with correlation coefficients of 0.999. The slopes were estimated to be 15 (35% T) and 4.2 (75% T). Each plot of migration time listed in Table I1 vs the base number was also linear (correlation coefficient = 0.999) for the poly(dA) and poly(A), and the slopes of the linear relationships were both 13 (3% T and 3% C). These slopes represent the speed of separation (basedmin), which strongly depends on the gel
migration time(min) Figure 7. plots of base number of poiy(A) versus migration time. Conditions: capillary 100-pm i.d., 375-pm o.d., 50-cm length, 30cm effecttve length; running buffer 0.1 M Tris-borate and 7 M urea, pH 8.6; gel matrix 1.5% C and (A)3% T, (0) 5 % T, and ( 0 )7 % T; field, 200 Vlcm; current, 11 FA.
composition. Such plots have been investigated20 by using polynucleotides in the chain length range from oligomer to 16Omer. Figure 7 illustrated that the linear relationship between the base number and the migration time was expanded to the higher chain length such as 450mer. In the present study we demonstrated that relative stable gel-filledcapillariesfree from bubbles were obtained by using the present method. The results in Figures 5 and 6 clearly illustrate that gel-filled capillaries prepared by this method show an ultrahigh resolution of the complex polynucleotide mixtures. Gel-filled capillaries possess the possibility of using C-PAGE as a useful tool for DNA sequencing. In addition, gel-filled capillaries are applicable to the rapid and highprecision check of purity of synthetic polynucleotides,which cannot be separated with single-base resolution by HPLC.7
ACKNOWLEDGMENT We appreciate Gerhard Schomburg a t Max-Planck Institute, Shigeru Terabe, and Takashi Manabe a t Himeji Institute of Technology for their helpful discussions on capillary gel electrophoresis. We acknowledge the assistance and discussions of H. Tanaka and s. Enomoto at Applied Biosystems Japan, Inc., and A. M. Chin, R. S. Dubrow, S. E. Morrison, J. E. Wiktorowicz,and J. C. Colburn at San Jose Operations, Applied Biosystems, Inc. RECEIVED for review November 4, 1991. Accepted March 6, 1992. Registry No. Poly(dA), 25191-20-2; poly(A), 24937-83-5; polyacrylamide copolymer, 25034-58-6. (20) Paulus, A.; Gassmann, E.;Field, M.J. Electrophoresis 1990,11, 702-708.
