Fiber Bundle Based Scanning Detection System for Automated DNA

Fiber Bundle Based Scanning Detection System for. Automated DNA Sequencing. Peter Trost and Andra´ s Guttman*. Genetic BioSystems, Inc., 10171 Pacifi...
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Anal. Chem. 1998, 70, 3930-3935

Fiber Bundle Based Scanning Detection System for Automated DNA Sequencing Peter Trost and Andra´s Guttman*

Genetic BioSystems, Inc., 10171 Pacific Mesa Boulevard, San Diego, California 92121

High-throughput DNA sequencing techniques are under rapid development currently, mainly triggered by the Human Genome Project. At the present time, slab gel based automated DNA sequencing is the standard procedure, utilizing fluorophore labeling and laser-induced fluorescence detection with scanning technology. In this paper, a novel, fiber-optic bundle based detection system is introduced, where a central illuminating fiber is used for the excitation of the electrophoretically separated fluorophore-labeled DNA sequencing fragments, along with several collecting fibers disposed around the illuminating fiber to collect the emitted fluorescent signal. As a model system, Cy5-labeled DNA sequencing fragments were separated on an ultrathin polyacrylamide slab gel and detected by the fiber bundle based laser-induced fluorescence detection system. A 640-nm diode laser was used to generate the illumination beam, and the emitted light collected by the fiber bundle was detected by a solidstate avalanche photodiode. DNA sequencing is a basic research tool in most molecular biology laboratories. The first powerful DNA sequencing techniques were reported in the late 1970s by Maxam and Gilbert (chemical method)1 and Sanger et al. (chain termination method).2 Both of these methods depend on subsequent analytical polyacrylamide gel electrophoresis separation to resolve oligonucleotides with one identical end and one end varying in length by a single nucleotide. The chain termination sequencing method proved to be simpler, quicker, and ready for easy automation. The technique employs radioactive labeling of DNA fragments, generated in four sets of sequencing reactions, terminated by the use of one of the corresponding dideoxynucleotides, and consequently size-separated by polyacrylamide gel electrophoresis. The detection of the separated radioactively labeled fragments was accomplished by autoradiography,3 and the sequence information was obtained by manually evaluating the patterns of the different lanes corresponding to the G-, A-, T-, and C-terminated reactions. Although this was the first viable method for large-scale DNA sequencing, it was very tedious and labor intensive. At the end (1) Maxam, A.M.; Gilbert, W. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 560564. (2) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467. (3) Sambrook, J.; Fritch, E. F.; Maniatis, T. Molecular Cloning, 2nd ed.; Cold Spring Harbor Laboratory Press: Plainview, NY, 1987.

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of the 1980s, several groups introduced the so-called automated DNA sequencing that refers to the automation on the base-calling process (i.e., reading of the sequence information) of the separated DNA sequencing fragments.4-6 These techniques all used either one or a set of four fluorophores to label the corresponding G, A, T, and C reactions, and the resulting fragments were separated by polyacrylamide slab gel electrophoresis. Fluorophore-labeled primers and dideoxynucleotide terminators have been utilized, respectively. During polyacrylamide gel electrophoresis separation, the fluorescently labeled DNA fragments are illuminated by a narrowband light source, focused into a small spot on the separation gel at the wavelength that is optimal to excite the fluorophore. This technique usually employs one or two laser line sources being focused onto the separation gel and a photomultiplier based detection mechanism on the same or the other side of the separation platform.7 A translation stage moves the detector head across the detection zone, providing continuous collection of the emitted fluorescent signal of the migrating bands.8 In highthroughput automated DNA sequencing, a thin gel, usually 0.0500.25 mm, is sandwiched between two glass plates. Fluorescently labeled DNA sequencing fragments are introduced into previously formed loading wells in order to form discrete lanes throughout the separation gel when the electric field is applied. Using crosslinked polyacrylamide sieving medium under denaturing conditions (e.g., 7 M urea), the migration velocity of the apparently identical mass-to-charge ratio DNA fragments is almost strictly dependent on their size (chain length); therefore, the labeled fragments propagate as discrete bands with different velocities.9 The fluorophore fluoresces light in the illuminating spot in an omnidirectional fashion, at a different wavelength from the illuminating light. The emitted light is then collected and focused onto a sensitive detection system. As mentioned above, the (4) Smith, L. M.; Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Connell, C. R.; Heiner, C.; Kent, S. B.; Hood, L. E. Nature 1986, 321, 674-679. (5) Prober, J. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F. W.; Robertson, C. W.; Zagursky, R. J.; Cocuzza, A. J.; Jensen, M. A.; Baumeister, K. Science 1987, 238, 336-341. (6) Ansorge, W.; Sproat, B. S.; Stegemenn, J.; Schwager, C. J. Biochem. Biophys. Methods 1986, 13, 315-317. (7) Adams, M. D. Nature 1994, 368, 474-475. (8) Ishino, Y.; Mineno, J.; Inoue, T.; Fujimiya, H.; Yamamoto, K.; Tamura, T.; Homma, M.; Tanaka, K.; Kato, I. Biotechniques 1992, 13, 936-943. (9) Freifelder, D. Physical Biochemistry; W. H. Freeman & Co.: New York, 1982. (10) Quesada, M. A.; Rye, H. S.; Gingrich, J. C.; Glazer, A. N.; Mathies, R. A. Biotechniques 1991, 10, 616-625. (11) Bashkin, J. S.; Bartosiewitz, M.; Roach, D.; Leong, J.; Barker, D.; Johnston, R. J. Capillary Electrophor. 1996, 2, 61-68. S0003-2700(98)00359-X CCC: $15.00

© 1998 American Chemical Society Published on Web 08/20/1998

excitation/detection system repeatedly scans across the gel, building an image of the migrating bands. Confocal detection systems are most widely used10 for scanning separation gel platforms. This method uses the very same lens set to focus the illuminating laser beam into the gel and to collect the fluoresced light emitted by the fluorophore-labeled sample. The illuminating beam from the laser diode is collimated and filtered through an interference filter. The filtered beam then propagates to a dichroic mirror that reflects the beam at a 90° angle. The reflected beam is then directed to a moving mirror and lens set, which moves along the beam, thus scanning the focused spot across the separating platform. The lens, which focuses the beam into the gel and collects the fluoresced light, usually has a large numerical aperture, so that a significant fraction of fluoresced light emitted by the sample is collected and formed into a well-collimated beam of fluoresced light. The fluoresced light beam propagates back along the path of the illuminating beam to the dichroic mirror, which is selected so that the emitted fluorescent light beam is transmitted through the mirror, propagating through a filter that rejects all light outside the spectra of the fluoresced light. A lens then focuses the emitted fluorescent light beam onto a detection device, such as a photomultiplier. Confocal systems are very efficient and relatively simple;11 however, alignment of the system is somewhat interdependent. Adjustment to any part may require that other parts of the system be adjusted as well, and, as Kheterpal et al.12 discuss, some reoptimization of the alignment is usually necessary on a daily basis. This paper describes an efficient fiber bundle based detection system with a simple alignment procedure, for detection of the fluorescent signal emitted by the migrating fluorophore-labeled DNA sequencing fragments. An optical apparatus is outlined for focusing the illuminating laser beam, for directing the focused excitation beam to the fluorescently labeled sample to induce fluorescent light emission, and for directing the emitted light to the collecting fiber for subsequent detection by an avalanche photodiode.

EXPERIMENTAL SECTION Materials. The Cy5-labeled M13 forward primer (-143) was synthesized by Genset, Inc. (La Jolla, CA). The ssM13mp18 DNA template, Thermo Sequense-DNA polymerase, deoxynulceotide triphosphates, and dideoxynulceotide triphosphates were from Amersham Life Sciences (Arlington Heights, IL). Acrylamide, N,N′-methylenebisacrylamide, TEMED, and ammonium persufate were from Research Organics, Inc. (Cleveland, OH). All other chemicals were from Sigma (St. Louis, MO). Fiber Bundle Based Detection System. Electrophoretic separation and detection of the Cy5-labeled DNA sequencing fragments were accomplished using a home-made system containing the components shown in Figure 1: (a) high-voltage EC6000P power supply (E-C Apparatus Co., Holbrook, NY), (b) 180 × 75 × 0.19 mm glass separation cassette with plexiglass buffer reservoirs, (c) translation stage (Distributed Motion Inc., Irvine, CA), (d) fiber-optic bundle, and (e) scanning lens set. The fiber (12) Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A.; Ju, J.; Ginther, C. L.; Sensabaugh, G. F.; Mathies, R. A. Electrophoresis 1996, 17, 18521859.

Figure 1. Block diagram of the DNA sequencing system. (a) Highvoltage power supply; (b) separation cassette; (c) translation stage; (d) fiber-optic bundle; (e) scanning lens set.

Figure 2. Fiber-optic bundle based laser-induced fluorescence detection system. See details in the Experimental Section.

bundle/scanning lens set based detection system of the unit contained the following components (Figure 2): (1) 640 nm (10 mW) red diode laser (DL-4038-021, Sanyo, Inc., Mountain View, CA), (2) aspheric lens (C330TM-B, Thorlabs, Newton, NJ), (3) narrow-band interference filter (F-12-640-24 mm, CVI Laser Corp., Albuquerque, NM), (4) lens (C260TM-B, Thorlabs), (5, 6, and 11) fiber bundle (SFM100/1120T, SPC210/230N, Fiberguide Ind., Sterling, NJ), (7 and 9) aspheric lenses (01 LAG 005, Melles Griot, Irvine, CA), (8) wide-band interference filter (682DF22, Omega Optical, Brattleboro, VT), (10) avalanche photodiode (C5460-01, Hamamatsu Co., Bridgewater, NJ), (12) spherical lens (KPx040, Newport, Co., Irvine, CA), (13) spatial filter (04PIP017, Melles Griot), (14) aspheric lens (350240, Geltech, Inc., Orlando, FL), and (15) glass separation platform. Apparatus. Ultrathin (0.19 mm) polyacrylamide gels (6% T/5% C) were cast (180 mm × 75 mm) in 100 mM Bis-Tris/Tricine (pH 7.2) buffer containing 2 mM EDTA-Na2 and 7 M urea, with a straight edge plastic comb inserted in the gel load end prior to polymerization. Sample introduction onto the separation gels was mediated by a 24-tab membrane loader (Genetic Biosystems Inc., San Diego, CA) by spotting 0.5-1 µL of the corresponding reaction mixtures onto the sampling tabs of the membrane.13 No sample purification or desalting was necessary in conjunction with membrane-mediated sample loading. The sample-spotted mem(13) Cassel, S.; Guttman, A. Electrophoresis 1998, 19, 1341-1346.

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brane was then gently inserted between the glass plates of the separation cassette in such a way that the membrane material came in direct contact with the separation gel. By the application of the electric field, the injection was completed, and the separation was started. Constant voltage of 1500 V was applied during the entire separation, generating 10-15 mA of current. The temperature of the separation platform was increasing during the separation from ambient to 40 °C due to Joule heating.14 Procedures. The sequencing reactions were accomplished according to the Amersham Life Sciences protocol. Single dyelabeled sequencing reactions were perfomed using Cy5-labeled M13 forward primer (-143) on a ssM13mp18 DNA template with Thermo Sequense-DNA polymerase in 20 cycles at 48 °C/30 s, and 95 °C/30 s, and terminated with the corresponding dideoxynulceotide triphosphates of ddATP, ddCTP, ddGTP, and ddTTP, respectively. Half a microliter of the reaction mixture (corresponding to 34 ng of DNA template) was injected onto the tabs of a membrane loader.13 The reactions were stopped by the addition of 3 µL of 83% formamide to each reaction mixture. It is important to note here that membrane loader-mediated sample injection does not require any purification of the sequencing reaction products, but samples were denatured at 95 °C for 2 min prior to spotting onto the membrane.

RESULTS AND DISCUSSION The system introduced in this paper was developed in our laboratory for the detection of Cy5-labeled DNA sequencing fragments separated on ultrathin polyacrylamide gels. Six sets of reactions were run in 24 lanes and scanned across the detection area, 12 cm from the injection point, by the fiber bundle based scanning detection device. Figure 1 shows the block diagram of the system, including the high-voltage power supply (a), the separation platform with built-in buffer reservoirs (b), the translation stage (c), and the fiber bundle/scanning lens set based detection system (d,e). As the fluorescently labeled DNA sequencing fragments migrate through the detection area of the separation gel, the lens set (e) that is connected to the fiber bundle (d) scans across the gel, by means of the translation stage, to collect the fluorescent light emitted by the labeled sample components. Light Source. Figure 2 shows the block diagram of the fiber bundle based detection system, including the solid-state laser diode (1) in the light emitting assembly (A), the avalanche photodiode (APD, 10) in the detecting assembly (B), and the fiberoptic bundle (11) with the scanning lens set (C) aimed to the detection area of the separation platform (15). The light beam of the laser diode (1) is collimated by an aspheric lens (2) with a focal length of 3.1 mm (Figure 2, section A). The collimated beam then passes through a narrow-band interference filter (3), which blocks wavelengths in the collimated input beam that are in the wavelength of fluoresced light. A second lens (4) is used with a focal length of 15.36 mm to focus the filtered, collimated input beam onto the face of the illuminating fiber. The longer focal length of the second lens, combined with the small diameter of the input light beam, allows the light beam to be launched into (14) Nelson, R. J.; Paulus, A.; Cohen, A. S.; Guttman, A.; Karger, B. L. J. Chromatogr. 1989, 480, 111-127.

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Figure 3. Cross-sectional view of the fiber-optic bundle. The excitation fiber in the center is surrounded by six collection fibers.

an illuminating fiber with a relatively small numerical aperture (0.12 or less). Detection System. In our system, an avalanche photodiode detects the emitted light, which is delivered to the diode by the collecting fibers at a relatively large numerical aperture (Figure 2, section B). The detection assembly includes a large aspherical lens (7) with 18-mm focal length and 24-mm diameter to collect the output light. The collimated beam passes through a 22-nmwide band interference filter (8) centered within the fluoresced light spectra. The interference filter is designed to heavily attenuate the primary laser wavelength. A second large aspherical lens, similar to (or the same as) the first lens (9), is used to focus the filtered, collimated beam onto a large area avalanche photodiode detector (10). Fiber Bundle. The fiber-optic bundle (11), as shown in Figure 2, includes an illuminating fiber (5) and several collecting fibers (6) disposed around the perimeter of the illuminating fiber (see details in Figure 3). In the depicted configuration, the illuminating fiber (5) is placed in the geometric center of the lens set (C), with the collecting fibers (6) located off the geometric center of the lens set. Figure 3 gives a cross-sectional view of the fiber-optic bundle used in our system, including six collecting fibers surrounding the perimeter of a single illuminating fiber of 0.1-mm diameter with a numerical aperture of 0.10. This relatively small numerical aperture (NA) of the illuminating fiber restricts the cone of an illuminating light beam output by the fiber to a fairly small angle. The cone angle of the excitation light beam (θ) is determined by the following equation:

NA ) sin θ/2

(1)

As eq 1 expresses, the numerical aperture, NA ) 0.10, provides a cone angle of 11.5°. The size of the cone is maintained at a relatively small angle, so that only the center-most area of the lens set, which provides minimal aberration, is used to process the excitation light beam. To minimize spherical aberration, it is also suggested that the cone angle be about 12° or less. The 2× lens set (Figure 2C) demagnifies the excitation light beam by a factor of 2, creating a spot of about 50 µm in diameter that illuminates the separating gel. The six collecting fibers (outer circles in Figure 3) are 230 µm in diameter, with a numerical aperture of 0.4 or greater. The configuration of the six collecting fibers as shown in Figure 3 reduces the dead space around the illuminating fiber. A black material, such as epoxy, is used to fill the dead space around and between the bundled optical fibers. Scanning Lens Set. The lens set of Figure 2C focuses the laser beam, to form the illuminating spot on the separation

lens (12). It is also shown that the collimated fluoresced beam has a circular cross-sectional area with a radius of ro that is larger than ri and forms an annular area encircling the cross-sectional area of the excitation beam. The ratio of the cross-sectional area to the annular area determines the collection efficiency (ηc ) into the inner illuminating fiber. The collection efficiency of the outer collecting fibers is determined by the following equation:

ηc ) 1 - πri2/πro2

Figure 4. Cross-sectional view taken along line D- - -D of Figure 2, section C, showing the cross-sectional areas of an illuminating beam and fluoresced light emitted by a sample at a lens located adjacent to a fiber-optic bundle.

platform (15), which excites the fluorophore-labeled DNA sequencing fragments while they migrate through the detection area during their separation by gel electrophoresis. The lens set includes a spherical lens (12), a spatial filter (13), and an aspherical lens (14). The 18-mm-focal length spherical-plano lens is located in front of the fiber-optic bundle (12). The spherical lens, together with the small numerical aperture of the illuminating fiber, forms a substantially collimated excitation beam with minimal aberrations. The spatial filter (13) defines the spot where the fluorescently labeled DNA is being detected in the gel. The 8-mm-focal length aspherical lens (14) is used to focus the collimated laser beam, to form the illuminating spot. A translation stage (Figure 1c) is used to move the lens set across the separating gel in the detection area. The fluorophore-labeled DNA sequencing fragments are excited by the focused laser beam. The omnidirectionally emitted fluorescent signal is then collected by the same lens set. The aspherical lens (14), with NA ) 0.5, forms the collected emitted light into a substantially collimated beam. The spatial filter (13) eliminates scattered light from outside of the detection area. The spherical lens (12) is matched in diameter to that of the spatial filter (13), and the aspherical lens (14) then forms the collimated fluorescing beam into a narrowing conical beam that is focused onto the collecting fibers (6) of the fiber bundle (11). When the excitation beam strikes on the spherical lens (12), the beam has a diameter of approximately ri ) 4 mm, as shown in Figure 4, which is a cross-sectional view along line D- - -D in section C of Figure 2. Thus, the collimated beam has a diameter of about 4 mm. The excitation beam is passed through the rest of the scanning lens set with minimal aberration, and a sharp focused beam is formed in the separation gel. The emitted fluoresced light is collected by the aspherical lens (14), which forms a collimated light beam with a diameter of approximately 8.7 mm. The emitted beam passes through the spatial filter with minimal aberrations and strikes the spherical lens (12). The majority of the rays passing through the outer portions of the spherical lens are aberrated onto the collecting fibers (6), rather than being focused onto the excitation fiber (5). The spherical lens (12) is selected such that the aberrations are significant outside of the 4.0-mm diameter of the excitation beam. As Figure 4 exhibits, the illuminating beam has a practically circular cross-sectional area, with a radius of ri, at the spherical

(2)

Substituting the corresponding values of ri (4 mm) and ro (8.68 mm) in eq 2, the resulting collection efficiency of the collecting fibers of Figure 4 is ηc ) 0.79. Other losses in efficiency of the fiber-optic bundle, besides losses due to imperfect collection efficiency, include packing fraction and Fresnel losses on the uncoated fibers themselves. Packing fraction is caused by the dead space between the fibers when they are arranged into a bundle, as shown in Figure 3. The packing fraction (PF) of this setup is approximately 0.78 in the active area of the bundle; that is, approximately 78% of the light is collected, and the remainder impinges on inactive areas of the bundle. The surface reflection (Fresnel) losses (Fl) on the interface surfaces of the fiber-optic bundle are typically 4% for each uncoated surface (in this instance, surface1 ) surface2 ) 0.04). Therefore, the total transmission (Tt) of the fiber bundle system is the product of the following:

Tt ) (ηc )(PF)(1 - Fl-surface1)(1 - Fl-surface2)

(3)

Thus, for the system described above, the total transmission of light collected by the objective lenses is 0.56 or 56%. The lost 44% of light is somewhat higher than that with a confocal system and resulted from the use of optical fibers. The comparable sensitivity of our fiber bundle based detection system to confocal systems suggests that this loss in fluorescent collection (which is higher than that of the confocal system) is probably compensated by an apparently higher signal-to-noise ratio. An in-house comparison between a confocal system and a fiber-optic bundle system indicated that the fiber-optic system had some advantage in S/N (data not shown). However, it is important to note that these differences are hard to quantify due to the low signal levels and various noise terms involved. The filters that we used in this system were six cavity interference filters representing the state of the art in this technology. With these filters, the attenuation of the laser line is, at best, 10-8 but probably closer to 10-6 in practice. While a holographic filter may perform much better and drop the laser background to a negligible level, the cost factor would be significant. Also, we suggest placing the illuminating fiber in the center of the fiber-optic bundle, so that the backreflected light is directed into the illuminating fiber and not into the detection leg of the system. This results in a significant noise reduction of the system that is supposed to compensate for the losses in overall light signal. Limit of Detection. The limit of detection (LOD) and the detection linearity for the presented fiber bundle based detection system were evaluated under regular electrophoresis separation conditions. Known amounts of Cy5-labeled DNA primer sets were Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

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injected onto the separation gel, and detection of the different dilutions was accomplished during electromigration. A linear response to Cy5-DNA conjugate concentration was found over the range of 5 × 10-11 M-2 × 10-9 M. The limit of detection, defined at the signal-to-noise ratio of 2, was found to be 5 × 10-11 M. This corresponds to 8.3 × 10-20 mol/pixel, and, considering the real separation conditions of our evaluation, the detection limit and linearity were comparable to others using unconjugated labeling dyes in organic solvent.11 Detection of DNA Sequencing Fragments during Electrophoresis Separation. The above-explained fiber bundle based scanning system was used for the detection of DNA sequencing fragments separated by ultrathin polyacrylamide slab gel electrophoresis. Figure 5 depicts a typical separation of cycle-sequencing reaction products of single-stranded M13mp18 template with Cy5labeled (-143) primer. Samples were injected without any prepurification using membrane-mediated loading technology. The 24 traces in Figure 5A correspond to six sets of sequencing reactions, using ddGTP, ddATP, ddTTP, and ddCTP termination for each reaction, injected onto the corresponding lanes of G, A, T, and C per set, respectively. The injected amount corresponds to 34 ng of DNA template per lane. As one can see, in this particular instance, all six sets depicted the same reaction. As an example, the corresponding four combined electropherograms representing the fragments resulted from the G, A, T, and C reactions for set number 4 is shown in Figure 5B, upper panel. Sequence information was obtained from this format starting 10 bases beyond the end of the primer, up to 350 bases (lower panel) in 2 h. The sequence was read and evaluated by an in-housewritten base-calling software. It is important to note here that all errors were identified as missing bases and are depicted in Figure 5B by lowercase underlined characters (lower panel). Three basecalling errors were found in the region of 10-300 bases, and five in the region of 10-350 bases, corresponding to 99.0% and 98.6% base-calling accuracy, respectively. The extension of the read length here is not detection system limited and can be extended by applying appropriate changes to the formulation of the separation matrix. SUMMARY This paper discussed the use of a fiber bundle based modular detection system, providing a significant advantage over confocal systems, where the alignment of each part of the system is interdependent; i.e., adjustment to any one part of the system requires adjustment of all other parts. The detection system presented here breaks the alignment process into three separate, mechanically unrelated modules linked together by the fiber bundle: (i) light-emitting assembly (A), including the diode laser, collimating lens, filter, and coupling lens; (ii) scanning lens set (C), including the collimating lens and aspherical objective lens; and (iii) detector assembly (B), including collimating lens, filter, coupling lens, and avalanche photodiode. These modules can be aligned independently; therefore, the adjustment to any one of the modules does not require an adjustment to any other parts in order to properly align the entire system. Please also note that our suggested setup does not require any routine daily alignment; actually, our two in-house-built systems have been used so far for almost 6 months without any realignment. The design presented here also increases the design flexibility, because any 3934 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998

Figure 5. Typical separation of cycle-sequencing reaction products by ultrathin polyacrylamide gel electrophoresis using fiber bundle based scanning detection system. The single-stranded M13mp18 template (34 ng/lane) was sequenced with the use of Cy5-labeled (-143) primer. Samples were injected by membrane-mediated loading technology without purification. Conditions: 1500 V constant separation voltage (i ) 10-14 mA), 7% T/5% C polyacrylamide gel in 100 mM Bis-Tris/Tricine, 2 mM EDTA, 7 M urea buffer (pH 7.2). Temperature at the end of the run, 40 °C. (A) Row trace data of the 24 separation lanes corresponding to six sets of sequencing reactions. ddGTP, ddATP, ddTTP, and ddCTP were used for termination of each sequencing reaction. The individual reactions were injected onto the corresponding lanes of G, A, T, and C on each set. (B) Combined electropherograms of the collected fluorescent signal of the G, A, T, and C lanes of set number 5 (upper panel) and the list of the called bases (lower panel). Base-calling errors are identified by lowercase underlined characters.

one of the three modules may be independently positioned without impact to any of the others. Because of the flexibility of the fiberoptic bundle, it may be arbitrarily routed, and modules A and B

may be positioned anywhere with respect to the scanning lens set, module C. The lens set should obviously be positioned in close proximity to the separation platform. Modern filter sets that permit extremely good detection limits using epi-illumination configuration are under evaluation in our laboratory. It is also important to note that the optical setup presented in this paper can be used for other applications besides DNA sequencing in which fluorescently labeled particles are being separated and scanned.

ACKNOWLEDGMENT The authors gratefully acknowledge the support of Enterprise Partners and Indosuez Ventures. The help of Nick Wilder and Swarna Ramanjulu is also greatly appreciated. Received for review March 30, 1998. Accepted July 13, 1998. AC980359U

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