Technical Notes Anal. Chem. 1996, 68, 2699-2704
A Capillary Array Gel Electrophoresis System Using Multiple Laser Focusing for DNA Sequencing Takashi Anazawa, Satoshi Takahashi, and Hideki Kambara*
Central Research Laboratory, Hitachi Ltd., Kokubunji, Tokyo 185, Japan
A very simple and highly sensitive capillary array gel electrophoresis system is constructed to analyze DNA fragments. On-column detection of DNA migration in a large number of gel-filled capillaries is carried out using side-entry laser irradiation and with a CCD camera, although it has been considered impossible because the irradiation laser is scattered strongly at the surfaces of the first few capillaries. By optimizing optical conditions, the laser beam can be focused repeatedly to irradiate all the capillaries held on a plate by working each capillary as a cylindrical convex lens. DNA sequencing samples migrating in 24 capillaries can simultaneously be analyzed with the system. The development of a high-throughput DNA analyzer is necessary for the high-speed DNA analysis required in the Human Genome Project.1-4 The capillary array gel electrophoresis technique is a promising method for this purpose. Several types of capillary array systems have been proposed so far.5-12 They include a scanning confocal fluorescence detection system,5-7 a multiple point irradiation system,8,9 and a multiple sheath-flow system.10,11 As a laser light is scattered at outer and inner capillary surfaces, one of the key points for successful implementation of a capillary array system is how to irradiate all the capillaries without producing a large background and with a sufficient laser strength to achieve a highly sensitive fluorescence detection. In a fluorescence detection system such as single-capillary gel electrophoresis systems,13-19 the detector is usually placed perpendicular to the irradiation laser light, because this geometry (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)
Wada, A. Nature 1987, 325, 771-772. Watson, J. D. Science 1990, 248, 44-49. Cantor, C. R. Science 1990, 248, 49-51. Hunkapiller, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. Science 1991, 254, 59-67. Mathies, R. A.; Huang, X. C. Nature 1992, 359, 167-169. Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967972. Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 21492154. Taylor, J. A.; Yeung, E. S. Anal. Chem. 1993, 65, 956-960. Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424-1431. Kambara, H.; Takahashi, S. Nature 1993, 361, 565-566. Takahashi, S.; Murakami, K.; Anazawa, T.; Kambara, H. Anal. Chem. 1994, 66, 1021-1026. Lu, X.; Yeung, E. S. Appl. Spectrosc. 1995, 49, 605-609. Luckey, J. A.; Drossman, H.; Kostichka, A. J.; Mead, D. A.; D’Cunha, J.; Norris, T. B.; Smith, L. M. Nucleic Acids Res. 1990, 18, 4417-4421.
S0003-2700(96)00183-7 CCC: $12.00
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minimizes the background signal due to light scattering. By using the side-entry laser irradiation technique, we have already employed this geometry for a conventional slab system20-22 and a capillary array system using multiple sheath-flow technique.10-11 If we can employ this geometry for a capillary array system using on-column laser irradiation, a very simple and highly sensitive system will be constructed. More recently Lu and Yeung have reported a system of this type,12 where nine capillaries were arranged on a plate in a closepack configuration and laser beam irradiation was from the side of the plane. They suppressed stray lights from the capillary surfaces by immersing the capillaries in water to roughly match the refractive index. We have independently developed a similar system by optimizing the optical conditions to irradiate efficiently all the capillaries placed in air as well as in water. In this paper, we demonstrate a very simple capillary array gel electrophoresis employing a multiple laser focusing technique. PRINCIPLE This study started from a question as to why a laser beam cannot irradiate a large number of capillaries simultaneously, while capillaries could act as cylindrical lenses to focus the laser beam. The simultaneous irradiation of the aligned capillaries might be possible if the focusing conditions are optimized. The reduction of laser beam intensity along the laser beam path during irradiation of capillaries is mainly due to the reflection and refraction of the beam at boundaries where the refractive index changes, which limits the number of capillaries in an array. Therefore, we estimated their effect on the reduction of the beam intensity. As refraction causes the intensity reduction more than reflection does, it was evaluated first. The refraction angle, ∆θ, (14) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; D’Cunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903. (15) Swerdlow, H.; Gesteland, R. Nucleic Acids Res. 1990, 18, 1415-1419. (16) Swerdlow, H.; Wu, S.; Harke, H. R.; Dovichi, N. J. J. Chromatogr. 1990, 516, 61-67. (17) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R.; Grey, R.; Wu, S.; Dovichi, N. J.; Fuller, C. Anal. Chem. 1991, 63, 2835-2841. (18) Guttman, A.; Cohen, A. S.; Heiger, D. N.; Karger, B. L. Anal. Chem. 1990, 62, 137-141. (19) Cohen, A. S.; Najarian, D. R.; Karger, B. L. J. Chromatogr. 1990, 516, 4960. (20) Kambara, H.; Nishikawa, T.; Katayama, Y.; Yamaguchi, T. Bio/Technology 1988, 6, 816-821. (21) Kambara, H.; Nagai, K.; Hayasaka, S. Bio/Technology 1991, 9, 648-651. (22) Kambara, H.; Nagai, K.; Kawamoto, K. Electrophoresis 1992, 13, 542-546.
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Figure 1. Cross section of a capillary and an incident laser beam path having infinitesimal diameter during transmission through the capillary. Refraction angle, ∆θ, of the laser beam by the capillary is illustrated. ∆θ is positive if the laser beam deviates counterclockwise. R and r are outer and inner diameters of the capillary; x is the distance from the center of the capillary to the incident laser beam; n1, n2, and n3 are the refractive indexes of outside, wall, and inside of the capillary, respectively.
Figure 2. Relation between refraction angle ∆θ versus R/r (ratio of outer diameter to inner diameter of a capillary), which are calculated from eq 1. Gel-filled capillary is placed (a) in water (n1 ) 1.33) and (b) in air (n1 ) 1.00); n2 ) 1.46 (quartz glass), n3 ) 1.36 (polyacrylamide gel), and x ) r/4.
which is defined as the angle between the incident laser beam direction and the laser beam direction after passing through a capillary as shown in Figure 1, is represented by eq 1, where R
{
(Rx ) + sin
∆θ ) 2 -sin-1
( )
-1
( )
n 1x n1x - sin-1 + n2R n2r
( )}
sin-1
n1x n3r
(1)
and r are the outer and inner diameters of a capillary, respectively; x is the distance from the center of the capillary to the incident laser beam, whose diameter has been assumed to be infinitesimal; and n1, n2, and n3 are the refractive indexes of the material outside of the capillary, the wall of the capillary, and the sieving medium inside of the capillary, respectively. The value of ∆θ is positive when the laser beam deviates counterclockwise as shown in Figure 1. ∆θ is always equal to zero when x ) 0, and the absolute 2700
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value of ∆θ increases with x when 0 e x < r/2. A capillary acts as a concave lens when ∆θ > 0, and it acts as a convex lens when ∆θ < 0. In general, the wall of a capillary plays the role of convex lens, and the inside of a capillary acts as a concave lens because of n1 < n2 > n3. The balance of these two opposite roles determines the overall optical characteristics of the capillary. When placed in water, capillaries with 2 < R/r act as concave lenses (∆θ > 0), as shown in Figure 2a. When placed in air, capillaries with 2 < R/r < 6, which are commercially available, act as convex lenses (∆θ < 0), as shown in Figure 2b. If multiple convex lenses are placed in tandem along one axis, the incident light can be refracted toward the axis repeatedly and transmitted through all the lenses without it escaping from the lens array. Similarly, a laser beam can irradiate many capillaries by being focused repeatedly by the capillaries which act as convex lenses. Laser beam paths along a capillary array are simulated, and the results are shown in Figure 3. Ten capillaries, with R ) 0.375 mm and r ) 0.075 mm (R/r ) 5), are placed on the same plane in a close-pack configuration. The incident laser beam (0.06 mm in diameter) irradiation was from the side of the capillary array plane. If the capillaries are placed in water, as shown in Figure 3a, the laser beam diverges and does not hit the gel inside effectively except for the first one because each capillary acts as a concave lens. However, if the capillaries are placed in air, as shown in Figure 3b, the laser beam passes the gel inside of all the capillaries, because each capillary acts as a convex lens to focus laser beam repeatedly. So, we called this irradiation technique “multiple laser focusing”. It could be possible to utilize 100% of the transmitted (refracted) laser beam when the capillary array is placed in air, assuming that its intensity does not decrease exponentially along the path because of reflection at every boundary. It is estimated by simulation that the incident laser power is reduced by 7% due to reflection by each single capillary, indicating transmittance of ∼93% per capillary (see Figure 5). Reflectance of incident laser power can be reduced to less than 1% per capillary by putting capillaries in water or some liquids of high refractive index as demonstrated by Lu and Yeung.12 However, multiple laser focusing does not work in these conditions, as shown in Figure 3a. Consequently, on the basis of above analysis, it is more desirable to place multiple capillaries in air than in water when polyacrylamide gel and quartz glass capillaries are used. EXPERIMENTAL SECTION Instrumentation. Figure 4 presents a schematic view of a capillary array assembly and a fluorescence detection system. From 10 to 24 fused-silica capillaries with 0.375-mm o.d. and 0.075mm i.d. (GL Science Inc., Tokyo, Japan) were arranged to make contact with each other (0.375-mm pitch) on a nonfluorescent glass slide (Matsunami Glass Ind., Ltd., Osaka, Japan). The glass slide was set up so that the detection window parts of the capillaries were vertical. A YAG laser (532 nm, 10 mW; Coherent, Santa Clara, CA) and a He-Ne laser (594 nm, 5 mW; Electro Optics, Boulder, CO) were mixed with a dichroic mirror to irradiate all the detection windows from the side of the capillary array plane. The mixed laser beam was focused by a lens (f 40 mm) to ∼0.06 mm on the first capillary in the array. The laser beam was adjusted to pass through the center of the capillaries. Both ends of the capillaries were separately immersed in two buffer vessels filled with 1 × TBE (89 mM Tris-boric acid, 2.5 mM EDTA, pH 8.3).
Figure 3. Computer simulation results of laser beam paths through a capillary arrary. Ten capillaries with 0.375-mm o.d. and 0.075-mm i.d. are arranged in the same plane and placed (a) in water and (b) in air. A laser beam of 0.06-mm diameter is introduced from the side of the capillary array; n1, n2, and n3 are the same as in Figure 2.
Figure 4. Schematics of a capillary array assembly and a fluorescence detection system. Detection window regions of 10 capillaries are touching together in the same plane and fixed on a glass slide. Fluorescence images are recorded by a two-dimensional cooled CCD camera with two lenses and a prism system between them. See text for details.
Fluorescence from DNA bands in each capillary was detected simultaneously with a two-dimensional cooled CCD camera (TEA/ CCD-1024EM/1, Princeton Instruments, Inc., Trenton, NJ) from a direction perpendicular to the capillary array plane as described in Figure 4. The CCD camera had two lenses attached (f 50 mm, F 0.95 and f 55 mm, F 1.2) and an image-splitting prism with optical filters between the lenses. The prism system produced four different color images of fluorescently-labeled DNA bands on the detector. The details of this multicolor detection method have been reported previously.11,21,23 Four bandpass filters of 554 ( 6, 567 ( 7, 618 ( 8, and 662 ( 20 nm (Omega Optical, Brattleboro, VT) coupled with two notch filters to cut off the excitation lights were used for detecting JOE, TAMRA (Applied Biosystems, Foster City, CA), Texas Red (Molecular Probes, Eugene, OR), and Cy5 (Biological Detection Systems, Inc., Pittsburgh, PA), respectively. In the case of only one-color detection, the Texas Red channel was used. The sampling period and the exposure time of the camera were 1.5 and 1.0 s, respectively. Each sampling data was sent to a workstation (Controller 382, Hewlett Packard, Palo Alto, CA) through a GPIB interface and recorded. Signals from a square area of 3 × 3 ) 9 pixels (0.081 × 0.081 mm2) in the detector (23) Kostichka, A. J.; Marchbanks, M. L.; Brumley, R. L., Jr.; Drossman, H.; Smith, L. M. Bio/Technology 1992, 10, 78-81.
Figure 5. Fluorescence intensity (top) and background intensity (bottom) versus the capillary position within the capillary array. Texas Red primer flowed continuously in the 10 open capillaries with 0.375mm o.d. and 0.075-mm i.d. Error bars are set as twice the standard deviation of the background intensity. Irradiation efficiency calculated from Figure 3b is superimposed and indicated by open circles.
were accumulated to give a signal from one capillary. The data analysis was performed with the Power Macintosh 7100/80AV. Preparation of Capillary Gel Columns. Capillaries were cut into 40 cm long segments with a detection window of ∼1 cm long at a position 30 cm from the inlet side. The detection windows were produced by burning off the polyimide coatings before filling the capillaries with gel. Any special treatment of the capillary inner surface for acrylamide adhesion was not carried out. An acrylamide solution of 4% T, 5% C in a 1 × TBE with 7 M urea was filtered with a 0.2-µm syringe filter and degassed under vacuum for 20 min. After the solution was cooled to 10 °C, N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
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Figure 6. DNA sequence spectrum from one capillary in a 10-capillary array. A-, C-, G-, and T-terminated fragments labeled with Texas Red (red), JOE (blue), TAMRA (green), and Cy5 (black), respectively, were electrophoresed at 100 V/cm. Each capillary was filled with 4% T, 5% C polyacrylamide gel in 1 × TBE and 7 M urea, with an effective length of 30 cm. Samples were injected for 2 s at 100 V/cm.
persulfate (APS) were added to the solution for a final concentration of 0.04% of the total volume. The solution was immediately injected into the capillaries with a syringe and allowed to polymerize to gel overnight at room temperature. Prior to the analysis of DNA, pre-electrophoresis was performed in 1 × TBE for 30 min at 100 V/cm. Samples were injected electrophoretically for 2 s, and then electrophoresis was performed at 100 V/cm. Preparation of DNA Sequencing Sample. A-, C-, G-, and T-terminated DNA sequencing fragments were separately produced by Sanger sequencing reactions with Texas Red-, JOE-, TAMRA-, and Cy5-labeled primer, respectively, and with a ∆Taq cycle sequencing reagent kit (Amersham International Plc, Buckinghamshire, UK). The reactions were carried out with 0.1 µg of M13mp18 single-stranded DNA template and 0.4 pmol of each fluorephore-labeled primer (18-mer oligonucleotide) in four tubes. After mixing four reaction products, ethanol precipitation was performed. The sample was resuspended in 4 µL of formamide. For single-color detection, only A-terminated DNA sequencing products labeled with Texas Red were used. The concentration and the produced amount of the sample were comparable to those used in conventional slab gel electrophoresis, although only one hundredth of it was required for the analysis in capillary gel electrophoresis. Prior to sample injection, the sample was heated at 90 °C for 2 min and then placed on ice. RESULTS AND DISCUSSION Sensitivity. Fluorescence intensity change along the irradiated region was obtained with 10 open capillaries to investigate the fluorescence detection sensitivity and determine the possible 2702 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996
maximum number of capillaries in the system by flowing buffer solution of 1 × TBE containing 10-9 M Texas Red-labeled primer. The buffer solution also contained 30% formamide to make its refractive index nearly equal to that of polyacrylamide gel (n3 ) 1.36). The refractive indexes of polyacrylamide in various concentrations have been reported by Chen et al.24 The refractive index of 4% T, 5% C polyacrylamide containing 7 M urea is ∼1.40. However, we have found that it decreased to nearly 1.36 after preelectrophoresis, probably because the urea concentration decreased or gel matrix conformation changed. Fluorescence intensity and background intensity of each capillary are indicated by the bar graphs in Figure 5, where each capillary was numbered from 1 to 10, with the capillary closest to the laser source being the first. The background was obtained without Texas Red. The fluorescence from the Texas Red-labeled primer was obtained by subtracting the background from the signal. The vertical axis unit is arbitrary, but the same scale is used for both graphs. Irradiation efficiency (the ratio of the irradiating laser power to the incident laser power) for each capillary calculated from Figure 3b is also presented in Figure 5 (top, right). The fluorescence intensity decreased gradually with the capillary number, which coincides with the simulated results. The fluorescence intensity from capillary 10 was 44.1% of that from capillary 1, which was a little bit smaller than 52.8% obtained with simulation. The average transmittance of laser power per capillary was estimated to be 91.3% from the experimental result, which coincides with the value (24) Chen, D.; Peterson, M. D.; Brumley, R. L. Jr.; Giddings, M. C.; Buxton, E. C.; Westphall, M.; Smith, L.; Smith, L. M. Anal. Chem. 1995, 67, 34053411.
Figure 7. Fluorescence ladder iamges using a 24-capillary array. A-terminated DNA sequencing fragments labeled with Texas Red were electrophoresed. All other conditions were the same as those in Figure 6. Image compensation was performed to try to equalize the signals from each capillary on the image.
93.1% estimated by simulation. It should be emphasized that a laser propagates through the capillary array at a very high efficiency of 91.3% per capillary because of the multiple laser focusing function of the capillary array. Background intensity also decreased gradually with capillary number. Error bars illustrated in Figure 5 indicate twice the standard deviation of background intensity, i.e., noise level for detection. The signal-to-noise ratios (S/N) was ∼1000 at capillary 1 and ∼700 at capillary 10, which correspond to a detection limit of ∼10-12 M for Texas Red in a capillary. Although this system was operated in four-color mode using an image-splitting prism with optical filters and only onequarter of the total fluorescence was collected per color channel, the sensitivity was comparable to the reported sensitivities with a single-capillary gel electrophoresis system,15 as well as a capillary array system reported by Lu and Yeung.12 This indicates that the present system has a very high sensitivity.6,8,9,11 Separation of DNA Sequencing Reaction Products. The DNA (M13mp18) sequencing result from one representative capillary in a 10-capillary array is shown in Figure 6, where the effective length of the capillaries was 30 cm and electric field strength was 100 V/cm. DNA bands were well separated and detected with a sufficient S/N. To demonstrate the highthroughput capability of the method, A-terminated DNA sequencing fragments labeled with Texas Red were electrophoresed using an array of 24 capillaries, where the other conditions were the same as in Figure 6. Figure 7 shows the ladder images of the electropherograms. Although the intensities of DNA bands changed from capillary to capillary as discussed previously, image compensation was performed to make the signals from each capillary nearly equal. The horizontal direction is the position along the capillary array, and the vertical direction is the migration time. Each capillary was numbered from 1 to 24, with the capillary
Figure 8. Electropherograms of first 10 capillaries using a 24capillary array. All other conditions were the same as those in Figure 6. The scales were adjusted as each spectrum gives almost the same peak heights.
closest to the laser source being the first. All tracks were wellresolved without cross talking. The first 10 of the electropherograms in a region from 370 to 520 bases are shown in Figure 8. The vertical axis unit is arbitrary but the same scale is applied to all the electropherograms. The numbers in Figure 8 represent the base numbers beyond the primer. The fluorescence intensities at the same base number decreased gradually with the capillary number in Figures 7 and 8. The fluorescence intensities from capillaries 10 and 20 were about half and a quarter of that from capillary 1, respectively, which is expected based on Figure 5. Here, the nonmonotonic decrease of the intensities along the capillary number was due to concentration fluctuation of the injected samples. All electropherograms show large S/N. The noise level was equal to ∼2 on the scale used in Figure 8. Although the S/N for capillary 10 was the lowest among the capillaries in Figure 8, it is 33 at 378 bases, which is still sufficient for sequencing. The migration speed and resolution of DNA bands in each capillary in Figures 7 and 8 were comparable to those obtained in a single-capillary gel electrophoresis system under the same conditions.25 The capillary to capillary variation in migration speeds of DNA fragments was very small. For example, in the 10 capillaries of Figure 8, the peak detection time of 378 bases was 121.9 ( 0.3 min, indicating that the relative standard deviation of the migration speed was ∼0.2%.6,9,11 This level of variation is mainly caused by the inhomogeneities in the gel matrix as well as variation in capillary lengths. The resolution of DNA band migration in each capillary was similar to each other in Figures 7 and 8. It is possible to separate two adjacent DNA bands with (25) Kamahori, M.; et al., submitted to Electrophoresis.
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one-base resolution over a DNA size of 500 bases. For example, the four peaks at 451-454 bases and the three peaks at 511-513 bases in Figure 8 are separated. CONCLUSION A simple and easy to handle capillary array gel electrophoresis system using multiple laser focusing is presented. It has no complex parts such as a laser scanning unit, a capillary array scanning unit, or a sheath-flow system. This system is suitable for non-cross-linked polyacrylamide or polymer gel as sieving medium because they could be replaced without taking out a capillary array from the detection region after every electrophoresis. Therefore the use of replaceable gel makes full automation of DNA sequencing possible.26-29 Another advantage of this system is its high sensitivity because there is no scanning and no dilution of samples in a buffer flow, which is very important for DNA analysis. We only showed 24 capillaries bundled in an array. However, more capillaries can (26) Ruiz-Martinez, M. C.; Berka, J.; Belenkii, A.; Foret, F.; Miller, A. W.; Karger, B. L. Anal. Chem. 1993, 65, 2851-2858. (27) Best, N.; Arriaga, E.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1994, 66, 4063-4067. (28) Zhang, J.; Fang, Y.; Hou, J. Y.; Ren, H. J.; Jiang, R.; Roos, P.; Dovichi, N. J. Anal. Chem. 1995, 67, 4589-4593. (29) Fung, E. N.; Yeung, E. S. Anal. Chem. 1995, 67, 1913-1919.
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be bundled to obtain a higher throughput by improving the detection sensitivity. It could be achieved by more concentrated sample, higher power laser, and/or capillaries of larger inner diameter. One possible way to further increase the irradiation efficiency is to use the transmitted light again by reflecting it with a mirror to reirradiate the capillary array along the reverse path. Another way is to use a laser beam divided with a beam splitter to create two beams to irradiate the capillary array from both ends, by which the number of capillaries could be doubled without losing sensitivity. ACKNOWLEDGMENT We thank Masao Kamahori, Takashi Yamada, Yoshinobu Kohara, and Hidetsugu Shimizu for their discussions and assistance on gel preparation and electrophoresis. We also thank Kiyoshi Fujimori and Yuki Manabe for the sample preparation. We also express our thanks to Dr. Rudian Chen for his comments on the paper. Received for review February 27, 1996. Accepted May 6, 1996.X AC9601831 X
Abstract published in Advance ACS Abstracts, June 15, 1996.
