Report
CAPILLARY ARRAY ELECTROPHORESIS
DNA SEQUENCING
T
he three-billion-base-pair human genome is the Holy Grail for human life. The sequential arrangement of the four bases—adenine (A), cytosine (C), guanine (G), and thymine (T)— defines the genetic code of an individual and is invaluable for understanding the chemical basis of life, genetic disease diagnosis and therapy, and individual identification. The goal of the Human Genome Project is to determine and decode this sequence information (1) Sequencing DNA especially three billion base pairs, is a challenging analytical measurement requiring ultimately part in three billion resolution! Therefore the Human Genome Project has driven the development of a variety of new analytical instruments methHHQ a n d rpntypnts for n u H e i c acid analvQiQ
gels in -80 min (2-4). Much Sequencing with a linked progress has been made in the past eight and sequencing read-lengths of single capillary is like years, more than 1000 bases can now be obtained in 80 min using replaceable linear using only one lane polyacrylamide gels (5). DNA sequencing with a single capilon a slab gel—youget lary (2-5) is analogous to using only one lane on a slab gel; despite the high speed a high-speed of the separation, the overall throughput still low. Thus, it is essential to run and separation, but the isdetect many capillaries simultaneously. In 1992, Mathies and Huang developed an overall throughput is approach called capillary array electro(CAE) for running multiple caplow. Running many phoresis illaries in parallel (6). They constructed a confocal fluorescence scanner and demcapillaries onstrated DNA sequencing in 25 parallel capillaries (7). A CAE instrument based simultaneously on this design which runs 96 capillaries (8) is being used by high-throughput becomes essential. genome centers and pharmaceutical com-
One method that is now becoming orominent for nroduction DNA sequencine- is capillarv array electrophoresis CE is an attractive technique for DNA resolution separations; increased separaanalysis because the narrow-bore, gelfilled capiilaries provide high-speed, high- tion efficiency; and automated gel and sample loading. The use of CE for DNA sequencing was first demonstrated in Indu Kheterpal 1990, when sequencing separations of Richard A. Mathies —350 bases were obtained on crossUniversity of California-Berkeley
panies for DNA sequencing and analysis The feasibility of CAE instruments for high-throughput further illustrated by the recent anwhole tiuman ffpnnme
shotgun seouencinc project (9) facilitated by CAE terbnolouv (10)
Analytical Chemistry News & Features, January 1, 1999 31 A
Report Detection systems In CAE, all the capillaries must be illuminated using acceptable laser powers and detected with high sensitivity. Numerous approaches have been developed to address this challenge using either scanning or imaging technologies. In scanning systems, either the capillaries or the detection system must be translated along the scan axis. A schematic of our four-color planar confocal fluorescence CAE scanner is shown in Figure 1 (11). This scanning design provides high sensitivity by using a high-numerical-aperture microscope objective, while the background fluorescence from the capillary and scattering from the surfaces are minimized through confocal sectioning. A laser power of only 3-4 mW at 488 nm from an argon ion laser is used for optical excitation. The capillary bundle is placed on a translation stage, which moves at 1 cm/s in a direction perpendicular to the direction of electrophoresis. The separation is sampled at a frequency of 2 Hz. High sensitivity and S/N are obtained, despite the sampling design of this system, because the optimum laser power is used (12) and because the repetitive scan rate permits the interrogation of the majority of labeled DNA molecules electrophoresing through the capillary. The fluorescence is divided into four detection channels by dichroic beamsplitters and band-pass filters and then focused through a pinhole to photomultipliers for detection
Alternative CAE instruments with imaging charge-coupled device (CCD) detection systems (shown in Figure 2) have used multiple-sheath flow (10), on-column illumination (13), and fiber-optic array illumination (14). In an imaging detection system, all capillaries must be illuminated simultaneously. The line focus (Figure 2a) and fiber-optic splitter (Figure 2b) methods require an intense laser for excitation. Furthermore, a large-diameter lens that is placed relatively distant from the array must be used to collect the fluoresresulting in a reduced numerical aperture Such an imaffin.21 system is more susceptible to laser scatter compared with confocal scanners because there is limited spatial filterinp- Imaginp" detection systems have no movine and can provide a 100% duty evele A schematic of the multiple-sheath flow system demonstrated by Kambara and co-
workers is shown in Figure 2c (10). The electrophoretic detection cell consists of three parts: a gel-filled capillary array, a gel-free optical cell, and a gel-free open capillary. DNA fragments from the separation capillaries are eluted into the sheath flow. These fragments flow out through the open capillaries. Fluorescence excitation is provided by an argon ion laser at 488 nm and a YAG laser at 532 nm allgned with the long axis of the flow cell. The fluorescence signals from all the capillaries in the sheath flow region are imaged onto a CCD. This system results in low background fluorescence and scatter because the sample is irradiated in a gel-free optical cell (10) An on-column detection system illustrating the design of Ueno and Yeung (13) is shown in Figure 2a. A 50-mW laser beam is directed through mirrors at a 45° angle and line-focused onto the capillary array by a plano-convex cylindrical lens. The illumi-
Using 96-capillary array scanner with automated sample and gel-matrix loading, the total run time for sequencing more than 500 bases is < 2 2. One might naively expect that serial scanning of 96 capillaries would result in more than a 96-fold reduction of the signal from each capillary. This expectation, however, ignores the interplay between the photodestruction limitations on S/N and the rapid scan rate in these instruments (12). The transit time for a 300-base DNA fragment moving at —100 um/s through the detection zone is ~8 s. Therefore at rate of 3 Hz —24 sweeps are made through the band Assuming that the detection zone is —20 um in diameter and the laser cower is saturatinc one discovers that —60y of molecules
Figure 1 . Schematic of a four-color planar confocal fluorescence CAE
in a a\vf*n band scanner. (Adapted w i t h permission from Ref. 11.) are interrogated by this scanner News & Features, January 1, 1999 3 2 A Analytical Chemistry
nated region of the capillary array is imaged onto the CCD camera through a 50mm-diameter lens. Simultaneous separation of 11DNA fragments in 100 capillaries has been accomplished using this setup (13). Detection limits in this system are dependent on the position of the capillary in the bundle because of the Gaussian distribution of laser intensity along the focused laser line. Also, off-axis illumination can produce higher scatter. An approach using fiber-optic illumination (Figure 2b) was developed by Quesada and Zhang (14). Fluorescent labels in a bundle of eight capillaries were illuminated, and the fluorescence was collected with two perpendicular sets of eight optical fibers. The data are imaged onto a spectrograph, which displays the full fluorescence spectrum of the eight capillaries in parallel. Low-numerical aperture illumination and high-numerical aperture collection fibers were used to achieve picomole detection limits. Although this performance is impressive it is necessary to develop more convenient capillaryfiber-opticaljunctions to aooly this approach to a larger number of capillaries In the systems just described, the goal is to scale up to 96 capillaries. Is it possible to run and detect even more capillaries simultaneously? We have recently developed a rotary confocal scanner (Figure 2d) that can easily break the 96-capillary barrier. In this system, a microscope objective and a mirror assembly revolve inside a ring of capillaries, exciting fluorophores and collecting fluorescence from each capillary. More than 1000 capillaries can be mounted around the 4-in. diameter ring and detected The opening art on p. 31A presents sequencing data from 128 capillaries run in parallel on the rotary scanner. The data from each capillary are presented as four adjacent lanes in four different colors, one for each base. Sequence data from base 280 in each capillary were aligned, and —15 min of data are presented here. The colors used for each base were C, blue; T, green; G, ,lack; and A, red. The famous pentet and quartet in the M13mpl8 sequence at bases 314-323 are clearly resolved as shown in the expanded views. In this run, the sample injection failed in 15 of the 128 capillaries but more than
Figure 2. Formats for CAE detection. (a) On-column line-focusing detection (13). (b) Fiber-optic array imaging detection. (Adapted with permission from Ref. 14.) (c) Multiple-sheath flow detection (10). (d) Rotary confocal scanning detection (46).
300 bases could easily be read in 80% of the remaining capillaries. This scanner at its full capacity of 1000 capillaries could provide a throughput of half a million raw bases per run, assuming 500-base read-lengths; the validation of this instrument and its extension to full capacity are currently underway. DNA sequencing DNA sequencing samples can be generated using various methods, including primer walking, unidirectional deletion, and shotgun sequencing (15,16). The shotgun sequencing approach is becoming the most popular method for large-scale DNA sequencing because it is cost-effective and most easily automated in a production setting. In one version of this method, DNA is extracted from cells and —50 kb DNA fragments are cloned into cosmids, which are then partially digested or sonicated to produce randomly cut fragments 2-4 kb long.
These fragments are purified and subcloned into vectors such as M13 or PUC19 and inserted into E. coli. After cell growtht the double- (ds) or single-stranded (ss) DNA is isolated and sequenced using the Sanger dideoxy chain termination method (17). Automated fluorescent versions of this method use either four differently labeled primers (18) or terminators (19). Figure 3 presents a schematic of the Sanger dideoxy method for DNA sequencing. Four reactions are set up, one for each dideoxynucleotide. The sample is heated to 95 °C to denature the template; the temperature is then reduced to 50-60 °C so that the 5' dye-labeled primer can anneal to the ss-template. The thermostable polymerase forms a complex between the template and the 3' end of the primer at 70 °C, and extends the primer by sequentially incorporating deoxynucleotides one base at a time. The labeled strand is extended until a
Analytical Chemistry News & Features, January 1, 1999 33 A
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dideoxynucleotide is incorporated. The ratio of deoxynucleotide to dideoxynucleotide is adjusted so that random terminations are obtained. This results in a distribution of DNA fragments extending to more than 500 bases. The incorporation rates of most currently used enzymes are a few hundred bases per second. At this point, the fragments can either be pooled and sequenced, or they can be carried through another 20-30 cycles on the same template to increase the quantity of sequencing fragments. Multiple cycles are performed by heating the reaction mixture again to 95 °C to denature the extended fragments from the template, followed by primer annealing and extension. The contents of all four tubes are pooled, and DNA is precipitated with ethanol to remove excess reagents and salts in order
to prepare it for electrophoretic injection and sequencing. Fluorescent labels The fluorophores first developed for labeled primer sequencing (18) did not have high molar absorbances at a single common excitation wavelength. To compensate for this deficiency, many CE sequencing detection systems used either two excitation wavelengths (3,10,20) or alternative base-coding strategies (7,20-22). The most powerful solution to this problem, however, is provided by improved fluorescent labels. Energy-transfer (EI) primers have been developed that this limitation in a simple and elegant (23 24) Figure 4 presents a schematic set of M13 ET primers. These prrmers contain a
Figure 3. Sanger dideoxy chain termination method and cycle sequencing. 34 A
Analytical Chemistry News & Features, January 1, 1999
common donor dye at the 5' end and an acceptor dye —8-10 nucleotides away. The presence of the common donor fluorophore allows the use of a single laser at 488 nm to efficiently excite all four fluorophore pairs. The excitation is nonradiatively ttansferred by rrsonance ET t t the acceptor dye, and therefore the fluorescence observed is largely that of the acceptor. The nomenclature used for these ET dyes is D-N-A, in which D is the donor, A is the acceptor, and N is the number of nucleotides between the donor and the acceptor. Some ET primers currently available denoted as F10F F10G F10T and F10R F (FAM) a fluorescein derivative is the common donor and the acceptors G (R6C) T (TAMRA) and Rfl?(Y50are all derivatives of rhodamine With 488 nm pvirita
primers pvhibit 9—14. x hitrher fluorescence single dye labeled nrimers (23) FT dve termina torsar no rnmmerciallv available from several sources. More recently we have demonstrated the advantages of cyanine dyes as the common donor in ET primers and generated a set denoted as C10R110, C10G, C10T, and C10R (Figure 4) (25). Because of the high molar absorbance of cyanine compared with FAM, fluorescence intensities as high as 24 x that of single dye-labeled primers have been obtained along with reduced spectral cross-talk. The use of rhodamine derivatives as the four acceptor fluorophores minimizes the electrophoretic mobility differences in the extended sequencing fragments (25). The higher signal intensities provided by ET primers result in longer read-lengths and higher base-calling accuracies and permit reduction in the of template (11 23-26) The use of ET primers for short tandem sizing short tanrlem repeat-based cancer diacrnnnew base coding strategies for DNA sequencing (29) have also been demonstrated Since the development of ET primers, several alternative ET primers and ET terminator dye labels have been developed based on the same principles (30-32). A detailed comparison of our ET dye primers with dipyrrometheneboron dye primers indicates that, for both sequencing and primer-based, fragment-sizing applications,
the ET primers developed by Hung and co-workers are superior (25,33). The linking of the ET-coupled dyes by a 4-aminomethylbenzoic acid or propargyl ethoxyamino groups, as in Big Dyes, facilitates synthesis. We have found that the use of such a short spacer can result in enhanced radiationless losses that degrade the overall signal strength compared with ET labels that use the optimum dye spacing (25). Sieving matrix Cross-linked polyacrylamide gels were initially used for capillary gel electrophoresis sequencing (2-4). A gel's separation performance is dependent on the pore structure, which, in turn, is dependent on the molecular weight and concentration of the monomer and cross-linking agents. Unfortunately, the irreproducibility of the polymerization, the instability of the resulting matrix, and the limited lifetime of these gels initially impeded the use of CE in production DNA sequencing. To automate CAE sequencing and to increase the lifetime of capillary columns, it was thus critical to develop a replaceable sieving matrix so that the column could be refilled and regenerated after each run. A replaceable sieving matrix should have low viscosity and high-separation efficiency. In most circumstances, a wall coating is required to minimize the electroosmotic flow (EOF). Numerous replaceable gels, including linear polyacrylamide (LPA) (34), polydimethyl-acrylamide (PDMA) (35), polyethylene oxide) (PEO) (36) hydroxyethyl cellulose (HEC) (37) and poly(vinylpyrrolidone) (PVP) (38) have been reported Low-viscosity polymer solutions (< 100 centipoise), such as PDMA and PVP, provide the single-base resolution required for DNA sequencing. Both polymers noncovalently coat the surface of capillaries to suppress EOF and capillary wall interactions. These polymers have moderate separation efficiencies, producing sequencing runs of 500-600 bases in —2 h. Moderate-viscosity polymers (10005000 cP) include PEO, HEC, and replaceable LPA PEO can be used with bare capillaries, but surface treatment with HC1 is required prior to each electrophoresis run (36). Sequencing separations to —300
Figure 4. Schematic of ET primers for M 1 3 . The signal strength enhancements over the single dye-labeled primers have been indicated. (Adapted with permission from Ref. 24.)
bases have been presented using a mixed PEO separation matrix (21). Both LPA and HEC require a capillary wall coating to suppress EOF and deliver similar separation efficiencies. Single-base T extension separations to —600 bases have been obtained in —70 min using HEC as the separation medium (37). Progress has been made in developing replaceable LPA sieving matrixes (5,34). The LPA polymerization process depends on the purity of the monomer, the oxygen content, and the temperature. Polyvinyl alcohol-coated capillaries were used along with long-chain 2% LPA gels to separate more than 1000 bases in —80 min at 60 °C. These gels can be easily pumped into capil-
laries using —100 psi and thus appear to have all the desired properties. We have evaluated replaceable LPA, HEC, and a mixture of PEO and HEC for DNA sequencing using identical separation conditions, samples, and detection systems. Replaceable LPA provided the longest read-length (-1000 bases) compared with —600 bases in HEC and < 300 bases in the HEC-PEO mixture in the shortest amount of time. Replaceable LPA should be very useful for large-scale sequencing projects that require long read-lengths and high separation efficiency. Long-sequence reads reduce the number of sequencing reactions needed thereby reducing the amount of reagents and eventually the cost
Analytical Chemistry News & Features, January 1, 1999 3 5 A
Report of DNA sequencing. Long-sequence reads also minimize the computational effort involved in assembly. Sample cleanup and injection
DNA sequencing reactions are performed in high-salt-concentration buffers (250 mM Tris-HCl, 12.5 mM MgCl2), which are necessary for polymerase to synthesize DNA. However, these high salt concentrations interfere with the electrokinetic injections widely used in CE. Thus, DNA sequencing samples are traditionally precipitated in ethanol followed by dissolution in formamide for injection. This time-consuming step leaves variable amounts of residual salt in the samples. Therefore, improved methods for sample cleanup are needed. Karger's group recently developed a method using poly(ether sulfone) ultrafiltration membranes and spin columns to remove template and salt from the Sanger
reaction products (39) This method increased the reproducibility sample injection and resulted in a 10-50-fold
Applications
It is essential to optimize and validate CAE systems and methods in high-throughput sequencing of real samples. Kheterpal and co-workers optimized the confocal CAE scanner, ET primers, and sequencing methods for mitochondrial (mt) DNA sequencing (11). Human mtDNA is used for evolutionary studies as well as for maternally linked individual identification. Each of the 12 motifs of the hypervariable region I (HVRI) of human mtDNA from a Sierra Leone population was sequenced with 99% accuracy. Figure 5 shows a representative electropherogram obtained from a CAE DNA sequencing run of mtDNA The figure demonstrates single-base resolution of the entire 500-bp fragment. This sequencing experiment provided a test for the CAE scanner and ET primers because of the different patterns of sequence variation present in the 12 motifs. Our planar CAE scanners, replaceable LPA matrix, and ET primers are also being used to sequence Anabaena genes involved in the biosynthesis of phycobiliproteins as a part of an undergraduate sequencing project at Berkeley. The sequencing and assembly of six fragments is in progress and more than 250,000 bases have been sequenced in the past academic year. The first twintron (an intron within an intron) in
cyanobacteria was recognized among these fragments. (An intron is an expressed sequence of nucleotides in a gene.) A 96-capillary version of our CAE scanner has been commercially developed and extensively optimized and validated for DNA sequencing. For example, a microbial whole genome shotgun sequencing project on the fungus Candida albicans has been completed using CAE with a replaceable LPA matrix and ET primers. The microbial genome templates sequenced as a function of read-length are presented in Figure 6. A majority of the read-lengths were —550 bases in 90 min. The overall readability, which is defined as the percentage of templates producing usable sequences, was 88%. These experiments demonstrate that CAE systems are capable of automated, high speed, and high-throughput DNA sequencing. Prospects
The full and optimized implementation of CAE technology will allow a major increase increase in injected samnle However it in sequencing capacity and speed. Neverwould be finite challentnncr to incornorare theless, CAE systems do have fundamental the centrifi ration and filtration tens int limitations. As the number of capillaries in high-throughput integrated systems. DNA the array increases, it becomes more diffi1 -c 4.4.1. J T r< I cult to manufacture and work with the arsample-purification methods using LC (40) ray. A second limitation is the inefficiency and size-exclusion chromatography (41, of injection. We believe that these and 42) have also been developed as a part of other problems can be integrated CAE systems. solved by the transition to However, the purification microfabricated CAE column lengths used in systems. both approaches are Microchip DNA se~30 cm, and thp required quencing of 200 bases in valvei tnd pumt would 10 min using cross-linked polyacrylamide gels was es o ooC s s first demonstrated by Woolley and Mathies (26). terns, e injection process Since then, the perforin CE ie very inefficient mance of these chips has improved significantly. sample needed ss contact More than 400 bases were me Capillaries is several separated in 15 min; onecolor sequencing separal l a l - l . l v / l l \.*» \Jt 1. fOj \)L II lei L tions (43) and four-color amount is actually enjected. separations of sequencing iNew mernosa ior emcienu fragments have recently Figure 5. Sequence of the light strand of the low-volume sample cleanup hypervariable region 1 of human mitochondrial DNA been performed in only and injection are needed to obtained using CAE and ET primers. 20 min on 7-cm-long colimprovt aucomation and The primers used for each base were: C, blue (F9F); T, green (F9J); umns and called to more sequce the cost ot CAE G, black (F9T); A, red (F9R). The total electrophoresis time is 130 min. than 600 bases (44). sequencing. (Adapted with permission from Ref. 11.) ,./Y\
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Analytical Chemistry News & Features, January 1, 1999
The quality of sequencing separations on microfabricated devices is rapidly approaching that obtained using conventional capillaries. This rapid advancement in performance has occurred because the cross-channel loading design permits the injection of small concentrated plugs with reduced salt and template interferences. Given this advance, together with the recent demonstration of a CAE system that can analyze up to 96 samples on a single 4-in. microplate (45) we anticipate that microfabricated systems will lead to even further improvement in the speed and throughput of CAE DNA sequencing in the future We thank Gary Wedemeyer for help preparing the figures and J. Ju of Incyte Pharmacauticals for providing Figure 6. Financial lupport was provided by the Office of Energy Research, the Office of Health and Environmental Research of the U.S. Dept of Energy under contract DE-FG-91ER61125, and by the National Institutes of Health 2R01HG01399. Additional support was kindly provided by Amersham Life Science and by an ATP grant through Aftymetrix and Molecular Dynamics.
Figure 6. Histogram depticting the number of sequencing runs performed on Candida albicans versus the read length on a 96-channel CAE scanner.
Indu Kheterpal is a graduate student at the University of California-Berkeley working on developing methods for DNA sequencing using CE. Richard A. Mathies, professor of chemistry at the University of CaliforniaBerkeley, focuses sis sesearch on developing instrumentation and methods for CAE and microfabricated devices for DNA analysis. Address correspondence to Mathies at Dept. of Chemistry, University of California, Berkeley, CA 94720 (
[email protected]).
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