`
`Anal. Cham. 1992, 64, 967-972
`temperatures above 200 °C. The cause of the discoloration
`is not known but resulted in the need to heat the vapor cell
`to successively higher temperatures each time it was used in
`order to totally absorb the laser scatter.
`Raman Spectra with the Hg Vapor Cell. The pulsed
`Raman spectra of ethanol with excitation at 253.65 nm is
`shown in Figures 3a (no Hg cell) and 3b (Hg cell, 250 °C, in
`place). Just as in the case of the Rb filter, the laser scatter
`was completely eliminated by absorption in the metal vapor.
`However, in this case, the resolution of the monochromator
`system (1-nm spectral bandpass, about 150 cm"1) was insuf-
`ficient to resolve the two closely spaced peaks at 1100 cm'1.
`In Figures 4a and 4b are shown the Raman spectra of a solid
`crystal of potassium nitrate with and without the use of the
`In Figure 4a the laser scatter is so dominant
`Hg vapor filter.
`that the Raman peaks at 1055 and 1350 cm'1 are obscured.
`In Figure 4b, these two peaks are clearly evident after at-
`tenuation of stray light by the Hg filter. The small peak at
`3600 cm'1 in Figure 4a is an artifact and does not appear in
`Figure 4b.
`
`ACKNOWLEDGMENT
`This research was supported by a grant from the National
`Institutes of Health: NIH-5-R01-GM 38434-04.
`REFERENCES
`(1) Pelletier, M. J. Appl. Spectrosc. 1990, 44, 1699.
`(2) Woodward, L. A. In Raman Spectroscopy : Theory and Practice : Vol.
`2, H. A. Szymanski, ed.; Plenum Press: New York, 1970; Vol. 2.
`Inc.: New York
`(3) Long, D. A. Raman Spectroscopy, McGraw-Hill,
`1977.
`(4) Devlin, G. E.; Davis, J. L.; Chase, L.; Geschwind, S. Appl. Phys. Lett.
`1971, 19, 138.
`(5) Schoen, P. E.; Jackson, D. A. J. Phys. E. 1972, 5, 519.
`(6) Wall, K. F.; Chang, R. K. Opt. Lett. 1986, 11, 493.
`(7) Grossman, J. J. Filter Spectrograph. U.S. Patent No. 3,865,490,
`1975.
`(8) Huang, Y.; Yu, P. Y. Rev. Scl. Instrum. 1988, 59, 190.
`(9) Gowers, C.; Hirsch, K.; Nielson, P.; Salzman, H. Appl. Opt. 1988, 27,
`3625.
`(10) Carlson, R. J.; Asher, S. A. Appl. Spectrosc. 1984, 38, 297.
`(11) Asher, S. A.; Flaugh, P. L.; Washinger, G. Spectroscopy 1986, 1, 26.
`(12) Flaugh, P. L.; O’Donnell, S. E.; Asher, S. A. Appl. Spectrosc. 1984,
`38, 847.
`(13) Carrabba, M. M.; Spencer, K. M.; Rich, C.; Rauh, D. Appl. Spectrosc.
`1990, 44, 1558.
`(14) Yang, B.; Morris, M. D.; Owen, H. Appl. Spectrosc. 1991, 45, 1533.
`(15) Chou, P. T.; Studer, S. L.; Martinez, M. L. Appl. Spectrosc. 1991, 45,
`513.
`(16) Hamaguchi, H.; Kamogawa, K. Appl. Spectrosc. 1986, 40, 564.
`(17) Puppels, G. J.; Huizinga, A.; Krabbe, H. W.; de Boer, H. A.; Gijsbers,
`G.; de Mul, F. F. M. Rev. Scl. Instrum. 1990, 61, 3709.
`(18) Smrthells, C. J. Metals Reference Book, Vol. II: Butterworths: Lon-
`don, 1962; p 655.
`(19) Radzig, A. A.; Smirnov, B. M. Reference Data on Atoms, Molecules
`and Ions: Springer-Verlag: Berlin, 1985.
`(20) Alkemade, C. Th. J.; Hollander, Tj.; Snellman, W.; Zeegers, P. J. Th.
`Metal Vapours In Flames: Pergamon Press: Oxford, 1982.
`
`Received for review November 4,1991. Accepted January
`27, 1992.
`
`CONCLUSIONS
`The Rb and Hg metal vapor filters were shown to be ef-
`It is clear
`fective in eliminating laser scatter Raman spectra.
`that further work must be done to study the stability of these
`filters with time at elevated temperatures and to examine their
`practical use with more optimized Raman spectrometers.
`When used in conjunction with a single mode diode laser,
`locked, for example, onto the cesium transition at 852.10 nm,
`and a compact, high-efficiency spectrometer with diode array
`or CCD detection, such filters may make possible more
`sen-
`sitive and certainly more compact Raman instrumentation.
`
`Capillary Array Electrophoresis Using Laser-Excited Confocal Fluorescence Detection
`
`Xiaohua C. Huang, Mark A. Quesada, and Richard A. Mathies*
`Department of Chemistry, University of California, Berkeley, California 94720
`INTRODUCTION
`large surface-to-volume ratio of the capillary channel, the use
`of thin capillary walls (50-150 Mm), and the high thermal
`Capillary electrophoresis (CE) has found widespread ap-
`conductivity of the wall material.1
`plication in analytical and biomedical research, and the scope
`Although CE provides rapid analysis, thus far the total
`and sophistication of CE is still rapidly advancing.1"5 Gel-filled
`throughput is not high because only one capillary can be
`capillaries have been employed for the rapid separation and
`analyzed at a time. Developing a method to increase the
`analysis of synthetic polynucleotides,6 DNA sequencing
`throughput of CE is a challenging and important task. One
`fragments,7"11 and DNA restriction fragments.12,13 Open-tube
`possible approach is to employ higher electric fields which
`capillary electrophoresis has attained subattomole detection
`would provide faster separations. Higher electric fields,
`levels in amino acid separations14 and proven its utility for
`however, often introduce overheating of the columns and
`the separation of proteins, viruses, and bacteria.15 Separation
`column failure. Another way to increase the throughput is
`of the optical isomers of dansyl amino acids has also been
`to run a large number of capillary separations in parallel. This
`successfully demonstrated.16 Micellar electrokinetic capillary
`approach uses an array of capillaries and is therefore called
`chromatography, isoelectric focusing, and on-column deriva-
`capillary array electrophoresis (CAE). CAE is potentially
`tization can all be performed on CE columns, demonstrating
`advantageous because the individual capillaries can be inde-
`the utility of capillary electrophoresis as an analytical and
`pendently manipulated at the inlet, thereby facilitating rapid,
`micropreparative tool.4,5
`In our approach, the
`parallel loading of multiple samples.
`The advantages of CE arise intrinsically from the use of
`capillaries are combined into a ribbon at the outlet for ease
`a small inside diameter (20-200 Mm) capillary. High electric
`In this way, a 2 order of
`of parallel, on-column detection.
`fields can be applied along small diameter fused-silica cap-
`magnitude increase in CE throughput should be achieved
`illaries without a significant increase in temperature. Since
`because hundreds of capillaries can be easily bundled for
`the electrophoretic velocity of the charged species is pro-
`detection.
`portional to the applied field, CE can achieve rapid, high-
`An important problem confronting capillary array elec-
`resolution separation. The reduced Joule heating in CE is
`trophoresis is detection. Since small amounts of sample are
`a result of the low current passing through the capillary, the
`injected in a capillary, a high-sensitivity detection system is
`indispensable. Laser-excited fluorescence has proven to be
`a sensitive detection method in capillary electrophoresis and
`
`* To whom correspondence and reprint requests should be ad-
`dressed.
`
`0003-2700/92/0364-0967$03.00/0
`
`© 1992 American Chemical Society
`
`Downloaded via UNIV OF VIRGINIA on November 6, 2018 at 18:18:25 (UTC).
`
`See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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`High Voltage Power Supply
`
`Laser light (1 mW, 488 nm at sample) is reflected by
`Figure 1. Schematic of the laser-excited, confocal fluorescence capillary array scanner.
`a long-pass dichroic beam splitter, passed through a 32X, N.A. 0.4 microscope objective, and brought to a focus within a 100-^m-i.d. capillary.
`The resulting fluorescence is collected by the objective, passed back through the beam splitter, and focused on a 400-um-diameter confocal
`pinhole. After the spatial filter, the emission is spectrally filtered and detected with a photomultiplier. The output is amplified, digitized, and then
`stored in a computer. A computer-controlled stage with 2.5-gm resolution is used to translate the capillary array past the optical system.
`
`in DNA sequencing.7'11,14,17'21 In most
`laser-excited fluores-
`cence detection schemes, the incident laser beam and the
`It is
`emitted fluorescence are perpendicular to each other.
`difficult to configure a system to detect an array of capillaries
`using this 90° geometry. We have recently introduced a la-
`ser-excited, confocal fluorescence gel scanner which provides
`enhanced detection of fluorescently labeled DNA in slab
`gels.22'26 This detection system utilizes an epi-illumination
`format where the laser is focused on the sample by a micro-
`scope objective and the emitted fluorescence is gathered by
`the same objective using a 180° geometry followed by confocal
`detection. This geometry is ideal for on-column detection of
`capillaries. Using confocal excitation and detection, the depth
`of field of the optical system is sufficiently small that only
`the interior of the capillary is probed. Background scattering
`and reflections from the capillary wall are rejected by the
`spatial filter and by spectroscopic filters. The utility of
`fluorescence microscope detection for CE has been recognized
`in previous studies utilizing single capillaries.21,28 We show
`can be used to detect an array of
`here that confocal scanners
`capillary columns with high sensitivity.
`EXPERIMENTAL SECTION
`Scanning Confocal Fluorescence Detection. Confocal
`fluorescence detection of capillary arrays is performed with the
`system shown in Figure 1. An argon ion laser (Model 2020,
`Spectra-Physics, Mountain View, CA) is used for excitation. The
`laser beam is expanded to 5-mm diameter, collimated, and then
`reflected through a 32X, N.A. 0.4 infinite conjugate objective (LD
`Plan-Achromat 440850, Carl Zeiss, West Germany) by a long-pass
`dichroic beam splitter (480DM, Omega Optical, Brattleboro, VT).
`This dichroic beam splitter reflects the excitation laser beam but
`transmits fluorescent light which is Stoke’s shifted to longer
`wavelengths. The objective focuses the exciting laser on the
`
`sample and gathers the fluorescence with very high collection
`efficiency. The use of an infinite conjugate objective permits
`vertical adjustment of the probe volume by translating the ob-
`jective with no significant perturbation of the optical alignment.
`The focused 1-mW beam at 488 nm had a 9-^m beam diameter
`and a 25->im confocal beam parameter. The fluorescence emission
`is passed back through the long-pass dichroic beam splitter to
`reduce laser interference and to separate the excitation and de-
`tection paths. The fluorescence is then focused by a 75-mm focal
`length lens on a 400-Mm pinhole which serves
`as the confocal
`spatial filter. The light passing through the pinhole is filtered
`by a 488-nm rejection band filter (488 RB filter, Omega Optical,
`Brattleboro, VT), a long-pass cutoff filter (Schott GG-495, Esco,
`Oakridge, NJ), and a fluorescence bandpass filter (530 DF60,
`Omega Optical, Brattleboro, VT) followed by detection with a
`cooled photomultiplier tube (RCA 31034A, Burle Industries,
`Lancaster, PA). The output of the phototube was amplified and
`filtered with a low-noise amplifier (SR560, Stanford Research
`Systems, Sunnyvale, CA), digitized with a 12-bit analog-to-digital
`board (DASH-16F, Metra-Byte, Taunton, MA), and stored in an
`IBM PS/2 computer.
`Data Acquisition and Imaging. During electrophoresis, the
`translation stage (Model 4000, Design Components, Franklin, MA)
`is programmed to continuously scan the capillary array back and
`forth at 20 mm/s in a direction perpendicular to the electro-
`phoresis direction. The image acquired in this way has two
`dimensions. One is a spatial dimension representing the physical
`image of the capillaries. The other is a temporal dimension
`proportional to the elapsed time. During a particular sweep,
`fluorescence data are sampled at 2000 Hz so the nominal image
`resolution is 10 /xm/pixel; thus, 10 pixels represent the interior
`100-Min width of any given capillary. The electronic low-pass filter
`was set at 300 Hz to provide high-frequency noise rejection while
`still passing the spatial frequencies required to define the 100-fun
`i.d. of the capillaries. An image of the migrating bands is built
`up as a function of time by accumulating periodic 1-s sweeps of
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`Figure 2. Exploded view of the capillary array holder. The holder was
`constructed from a ’/4-in.-thick 5- X 5-cm aluminum base plate and
`a V1(-in.-thlck 2- X 4-cm cover plate. The plates were held together
`by four screws, and there was a matching 4- X 20-mm slot milled in
`each. The capillaries were sandwiched between the base and cover
`such that the exposed windows could be viewed through the slot.
`
`the exposed region of the capillaries. The transit time of the
`migrating DNA past the probe region, under the conditions em-
`ployed here, ranges from approximately 10 s for the low molecular
`weight fragments (40-50 mers) to 14 s for the higher molecular
`weight fragments (380-390 mers). With l-s repeat cycles, this
`gives 10-14 data points for each band. The computer controls
`the translation stage and displays the acquired image in real time.
`Image processing was performed with the NIH program, Image
`1.29, and a commercial image processing package, Canvas. The
`image data were reduced to a one-dimensional line plot or elec-
`the width of each
`tropherogram by averaging the pixels across
`lane.
`Preparation of Capillary Gel Columns. Zero-cross-linked
`poly(acrylamide) gel-filled capillaries were prepared using a
`modified version of the procedure described by Cohen et al.6-7 A
`3-mm-wide detection window was produced in each lOO-jim-i.
`d./200-/xm-o.d. fused-silica capillary (Polymicro Technologies,
`Phoenix, AZ) by burning off the polyimide coating with a hot wire.
`The window was burned ~25 cm from the inlet side of the 40-
`cm-long capillary. The inner wall of the capillaries was
`then
`treated overnight with a bifunctional reagent, [y-(methacryl-
`oxy)propyl]trimethoxysilane to prepare the walls for acrylamide
`adhesion.6 Freshly-made acrylamide gel solution (9% T, 0% C)
`in a 1 x TBE buffer (Tris-boric acid-EDTA) with 7 M urea was
`filtered with an 0.2-/im syringe filter and degassed under vacuum
`for about 1 h. 10% TEMED (tetraethylmethylenediamine) and
`10% APS (ammonium persulfate) solution were added to the gel
`solution at a final concentration of approximately 0.03%. The
`solution was immediately vacuum siphoned into the capillaries
`and then allowed to polymerize overnight in a cold room. Prior
`to use, both ends of the column were trimmed by about 1 cm and
`
`Image obtained by scanning a four-capillary array. Fluor-
`Figure 3.
`escent primer-labeled M13mp18 DNA “G" fragments were
`loaded In
`100-jzm-i.d. capillaries and then electrophoresed at 240 V/cm (9%
`T, 0% C, 90 mM Trls-borate). The capillary length from the inlet to
`the scan window was 24 cm and the total length was 38 cm. Elec-
`trokinetic sample Injection was performed for 10 s at 9 kV. Each pixel
`represents a 10 pm X 1 s portion of spatial and temporal information.
`then pre-electrophoresed for 30-60 min at 7 kV. The 9% T, 0%
`C gels were sufficiently stable that at least four consecutive DNA
`sequencing separations could be performed.
`Capillary Array Assembly. The capillary array was sand-
`wiched in a capillary holder that is mounted on the translation
`stage. The capillary holder shown in Figure 2 served the dual
`purpose of (1) uniformly constraining each capillary in the array
`to an identical height above the top of the translation stage and
`(2) exposing a small window through which the confocal zone
`probed the capillary interior. Constraining the capillaries to the
`same plane was critical for achieving uniform detection sensitivity
`from each capillary.
`Generation of DNA Sample. Chain-terminated M13mpl8
`DNA fragments were generated using a Sequenase 2.0 sequencing
`kit (United States Biochemical Corp., Cleveland, OH) and a
`fluorescein-tagged primer (FAM, Applied Biosystems, Foster City,
`CA). The detailed procedure has been published elsewhere.26
`Briefly, about 1 pmol of the primer and single-stranded M13mpl8
`DNA were heated to 65 ®C for 3 min and then allowed to cool
`the sequencing extension
`(annealing reaction). Meanwhile,
`mixture was added into a centrifuge tube followed by addition
`of the dideoxy-termination mixture. When the temperature of
`the annealing reaction mixture dropped below 30 °C, a combi-
`
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`1 21
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`189
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`269/270
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`31 4/31 5
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`473
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`B
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`D
`
`5000
`Figure 4. Electropherograms of DNA separation on the four-capillary array. The data in Figure 3 were reduced to a one-dimensional line plot
`by averaging the pixels across the width of each capillary. The base sequence can be read with high signal-to-noise out to greater than 500
`bases beyond the primer. The most intense peaks plateau at the same value because of electronic saturation of the amplifier.
`
`8
`
`si
`
`Figure 5. Expanded view of the Indicated regions from the four-capillary array electropherograms In Figure 4.
`nation of the labeling mixture and diluted enzyme (Sequenase
`other 5 min at 37 °C.
`Instead of adding stop solution, ethanol
`2.0) were added, and the mixture was incubated for 5 min at room
`precipitation was immediately used to terminate the reaction and
`temperature. This mixture was
`the DNA sequencing sample. The high concentration of
`then transferred to the tube
`recover
`having the termination mixture and allowed to incubate for an-
`conductive ions present in the DNA sequencing sample after the
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`termination step would reduce the amount of DNA that can be
`loaded into each capillary by electrokinetic injection. To coun-
`teract this effect, ethanol precipitation was performed on all DNA
`of 80% (v/v) formamide
`samples followed by resuspension in 6
`to give a concentration about 10-fold higher than that used in
`slab gels. The sample was heated at 90 °C for 3 min to ensure
`denaturation and then placed on ice until sample injection.
`Sample Introduction.
`The DNA sequencing sample was
`placed in a 500-centrifuge tube for electrokinetic injection.
`The same electric field strength (240 V/cm) used during separation
`was also applied during sample injection. The typical injection
`time was 10 s. After injection, the inlets of the capillaries were
`removed from the centrifuge tube and placed into a reservoir filled
`with fresh buffer.
`RESULTS AND DISCUSSION
`Figure 3 presents an image obtained from on-line confocal
`scanning of a four-capillary array during electrophoresis of
`a mixture of DNA sequencing fragments. The horizontal
`direction is the physical dimension representing the geometric
`arrangement of the capillary array while the vertical direction
`is temporal representing the passage of fluorescent DNA
`fragments through the detection window. For lane-to-lane
`comparison, identical samples of “G" base DNA fragments
`were simultaneously electrokinetically injected into each ca-
`pillary. The overall elapsed data acquisition time is ~80 min
`after passage of the primer. An expanded region of the image
`is presented in Figure 3. The bands in all four lanes are well
`resolved, and the resolution extends throughout the se-
`quencing run with sufficient signal-to-noise to detect bands
`than 500 bases beyond the primer.
`more
`Figures 4 and 5 present line plots of the DNA signal inte-
`the width of each capillary. A signal-to-noise
`grated across
`ratio of approximately 20 is observed out to base 385 (~65
`min) and bands are detected out to base 500 with the present
`experimental conditions. The number of theoretical plates
`is >1.9 X 106 (at base 385) over
`a 24-cm effective column
`length.
`A comparison was made between the signal-to-noise ratio
`obtained in the scanning mode and the case where the system
`is focused in the center of a single stationary capillary. The
`latter approach is analogous to traditional on-column detection
`from a single capillary. The sensitivity limits extrapolated
`for the scanning mode were found to be ~2 X 1CT12 M (S/N
`= 3) by flowing 1 X 10'11 M fluorescein through an open
`capillary. The sensitivity limits for the stationary mode were
`found to be ~1 X 10'12 M indicating that scanning does not
`seriously degrade the signal-to-noise ratio. These detection
`limits are at least as good as those reported from single cap-
`illaries using the conventional 90° detection geometry.10 The
`background from the gel-filled capillaries was ~2.6 times (n
`= 4) higher than that from a capillary filled with just TBE
`buffer. Thus, the presence of the gel increased the background
`noise by a factor of ~ 1.6.
`This work indicates that the overall throughput perform-
`ance of CAE can be very high. Figures 3-5 show that satis-
`factory sequencing information is obtained out to 500 bases
`for each of four capillaries in less than 2 h. An important issue
`is determining how feasible it is to increase the size of our
`capillary arrays. Figure 6 presents the results of a DNA
`sequencing fragment separation performed using an array of
`24 capillaries. The resolution, signal-to-noise, and background
`are uniformly good for all the capillaries in the array, dem-
`onstrating that extrapolation to larger numbers of capillaries
`is feasible. The limiting throughput of CAE using our system
`depends upon the total number of capillaries, N, that can be
`scanned. The equation, N = vT/2D, defines how N depends
`on the scan speed (a), the scan repetition period (T), and the
`capillary outside diameter (D). For example, we can run 100
`200-/im-o.d. capillaries using a scan rate of 4 cm/s and a l-s
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`ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 • 971
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`10
`
`15
`
`20
`
`24
`
`26
`
`105
`
`- 248
`
`Image obtained by scanning a 24-capllary array. The DNA
`Figure 6.
`sample consisted of fluorescent primer-labeled M13mp18 T-fragments.
`All other conditions were
`the same as those in Figure 3.
`
`Increasing the array size would require
`scan repetition period.
`(1) an increase in the scan speed, (2) the use of smaller o.d.
`capillaries, and (3) an increase in the scan repetition period
`which would reduce the temporal resolution of the electro-
`phoretic separation. Since reliable scan systems have velocities
`up to 10 cm/s and capillaries with o.d.’s of 150 nm are com-
`mercially available, a limit of approximately 330 capillar-
`ies/array can be projected assuming a l-s scan repetition
`period.
`Finally, it should be noted that there is a difference in the
`migration time of a given DNA band from capillary-to-ca-
`pillary. This may be caused by inhomogeneities of the gel
`matrix or the presence of local nonuniform variations in the
`It has previously been estimated that
`electric field strength.
`there is a 5% variation in migration time between identical
`samples on different gel columns.4 Table I lists the migration
`time for selected DNA fragments from each capillary in the
`four-capillary array, and a similar variation in migration time
`is observed.
`The velocity shift of the DNA bands from capillary-to-ca-
`pillary may preclude sequencing DNA with CAE using a single
`fluorophore and four different capillaries, one for each base.
`For DNA sequencing, the present apparatus must be ex-
`panded to a multicolor detection system to sequence all four
`
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`Table I. Migration Time of Selected DNA Fragments in
`the Four-Capillary Array
`migration time (s)
`column 2
`column 3
`
`column 4 % CV
`
`base
`number
`
`column 1
`
`87
`144
`185
`232
`285
`335
`385
`
`1308
`1734
`2069
`2594
`3129
`3621
`4168
`
`1286
`1729
`2065
`2454
`2891
`3300
`3679
`
`1283
`1808
`2166
`2448
`2884
`3287
`3658
`
`1337
`1764
`2112
`2583
`3079
`3499
`3902
`
`1.9
`2.2
`2.5
`3.2
`4.2
`4.7
`6.2
`
`bases in a single capillary. Four-color detection schemes have
`been developed for single capillaries8 and for slab gels.19 We
`have recently introduced a multicolor confocal fluorescence
`scanner and used it for DNA detection in slab gels.26 The
`successful application of this multicolor fluorescence scanner
`to the detection of capillary arrays should provide a valuable
`new sequencing technology.
`In summary, we have demonstrated that it is possible to
`perform high-sensitivity fluorescence detection of capillary
`arrays by using a confocal fluorescence scanner. The most
`important advantage of this approach is that multiple samples
`can be easily loaded and rapidly separated in parallel followed
`by high-sensitivity detection. The use of capillary arrays
`potentially resolves the throughput problems that limit the
`utility of CE in, for example, DNA sequencing.29 In addition,
`CAE provides an opportunity for the large-scale optimization
`of analytical separations. Commercially made capillary arrays
`could be constructed for large-scale parallel sample intro-
`duction. CAE should be a valuable new technique for rapid,
`parallel separation and analysis.
`ACKNOWLEDGMENT
`We thank Alexander Glazer
`for valuable discussions
`throughout the course of this work and Jiun-Wei Chen for
`helpful suggestions on the preparation of non-cross-linked
`poly(acrylamide) gel columns and DNA sequencing samples.
`This research was supported by the Director, Office of Energy
`Research, Office of Health and Environmental Research of
`the U.S. Department of Energy, under Contract DE-FG-
`91ER61125. X.H. was supported as a DOE Human Genome
`Distinguished Postdoctoral Fellow during the course of this
`research.
`
`REFERENCES
`(1) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272.
`(2) Gordon, M. J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science
`1988, 242, 224-228.
`(3) Ewing, A. G.; Wallingford, R. A.; Oteflrowicz, T. M. Anal. Chem. 1989,
`61, 292A-303A.
`(4) Karger, B. L.; Cohen, A. S.; Guttman, A. J. Chromatogr. 1989, 492,
`585-614.
`(5) Kuhr, W. G. Anal. Chem. 1990, 62, 403R-414R.
`(6) Cohen, A. S.; Najarian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.;
`Karger, B. L. Proc. Natl. Acad. Scl. U.S.A. 1988, 85, 9660-9663.
`(7) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990, 516,
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`Agilent Exhibit 1266
`Page 6 of 6
`
`
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