Ultrahigh-speed DNA sequencing
`using capillary array electrophoresis
`chips
`
`Adam T. Woolley, Richard A. Mathies
`
`Adam T. Woolley, Richard A. Mathies, "Ultrahigh-speed DNA sequencing
`using capillary array electrophoresis chips," Proc. SPIE 2386, Ultrasensitive
`Instrumentation for DNA Sequencing and Biochemical Diagnostics, (3 April
`1995); doi: 10.1117/12.206006
`Event: Photonics West '95, 1995, San Jose, CA, United States
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`Ultra-High-Speed DNA Sequencing Using Capillary Array
`Electrophoresis Chips
`
`Adam T. Woolley and Richard A. Mathies
`
`Chemistry Department, University of California
`Berkeley, CA 94720
`
`Keywords: microfabrication, DNA sequencing, allelic fragment sizing,
`restriction fragment sizing, confocal fluorescence detection, biosensors
`
`ABSTRACT
`
`Capillary electrophoresis arrays have been fabricated on planar glass
`substrates using photolithographic masking and chemical etching. The
`photolithographically defined channel patterns were first etched in a
`glass substrate, and then capillaries were formed by thermally bonding
`the etched substrate to a second glass slide. High-speed separations of
`restriction fragment digests were performed on these chips in under 1
`minute. Multiple separations in the same channel demonstrated excellent
`reproducibility. Rapid genetic typing of short tandem repeats on the
`HUMTHO1 locus have also been performed.
`Finally, separations with
`single-base resolution have been successful, indicating that these
`microfabricated devices will also be useful for high-speed DNA
`sequencing.
`
`1. INTRODUCTION
`
`Capillary electrophoresis (CE) is a powerful method for DNA analysis,
`including sequencing, forensic identification, polymerase chain reaction
`(PCR) product analysis, and restriction fragment sizing.1'2 Our group3
`first developed the technique of capillary array electrophoresis (CAE)
`in which separations are performed on an array of parallel silica
`capillaries, and we demonstrated the use of CAE for DNA sequencing,4'5
`restriction fragment sizing,6 and short tandem repeat (STR) analysis.7
`CAE combines the fast separation times of CE with the ability to analyze
`many samples in parallel. CAE increases the information density in
`electrophoresis by miniaturizing the 'lane" dimension to 100 .tm.
`The
`further miniaturization of electrophoretic separations to increase the
`number of lanes, as well as the speed and throughput will be essential
`in helping to meet the needs of the Human Genome Project.8'9 We have
`therefore pursued the use of microfabrication techniques used in the
`manufacture of integrated circuits for the production of higher density
`capillary arrays.
`
`Recently, several groups have performed capillary zone
`electrophoresis separations of fluorescent dyes10'11 and fluorescently-
`labeled amino acids12'13 on individual CE devices fabricated on glass
`chips. Our work has focused on the microfabrication of multiple
`
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`separation capillaries on glass chips, and the performance of high-
`resolution separations of DNA restriction fragments and PCR products on
`these chips.14 However, it remains to be seen whether DNA sequencing on
`microfabricated chips is possible.
`
`We describe here the use of photolithography and chemical etching to
`make large numbers of CE channels in a single substrate.
`We have
`developed procedures for filling these channels with hydroxyethyl
`cellulose (HEC) and polyacrylamide separation matrices, and have
`performed high-speed, high-resolution double-stranded DNA separations in
`these channels with excellent reproducibility. Furthermore, we have
`performed high-speed separations of STR alleles of the HUNTHO1 locus for
`forensic identification. Finally, we have demonstrated the feasibility
`C)f DNA sequencing on chips by performing sequencing separations with
`single-base resolution. The demonstration of high-speed DNA separations
`C)fl microfabricated CAE chips establishes the feasibility of
`manufacturing integrated devices for electrophoretic DNA analysis.
`
`2. CHIP FABRICATION
`
`Electrophoresis chips were made by bonding a chemically etched glass
`bottom substrate to a drilled glass top substrate to form capillaries.
`The etched pieces were fabricated by coating a glass substrate with a
`photoresist film and then transferring the channel pattern to the film
`by exposure to till radiation through a patterning mask. The exposed
`areas on the film were dissolved and the remaining photoresist was
`hardened by heating. The uncovered portions of glass were chemically
`etched and the photoresist film was then stripped off.
`The etched
`substrate was thermally bonded to the top glass plate which had access
`holes drilled through it.
`
`Figure 1A shows the laser-excited, confocal-fluorescence detection
`system and the dimensions and layout of the CAE chips. The detection
`system has been described elsewhere.14 On the chips, the injection
`channels connect reservoirs 1 and 3, and the separation channels connect
`reservoirs 2 and 4.
`Sample injection is performed by applying an
`electric field between reservoirs 1 and 3 with reservoir 1 at ground;
`electrophoresis is performed by applying a field between reservoirs 2
`and 4 with reservoir 2 at ground.
`Figure lB shows an electron
`micrograph of reservoir 3 and the intersection of an injection and
`separation channel; Figure 1C shows a higher magnification image of such
`an intersection. The well-defined channel pattern and the sloping side
`walls are clearly visible.
`
`3. DNA FRAGMENT SIZING
`
`DNA fragment sizing is a technique of rapidly expanding importance and
`utility. DNA fragment sizing is used for restriction fragment and PCR
`product sizing, as well as for the forensic techniques of restriction
`f:ragment length polymorphism and allelic typing of STR sequences.
`
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`A photomultiplier tube
`confocal pinhole
`focusing lens
`bandpass filter
`dichroic beam splitter
`
`40 X objective
`electrophoresis chip
`
`Figure 1. A: Schematic of the capillary array electrophoresis chip
`B: Low-
`and laser-excited, confocal-fluorescence detection system.
`magnification electron micrograph of an injection reservoir and the
`intersection of an injection channel with a separation channel.
`C: High-magnification electron micrograph of the intersection of an
`injection and separation channel.
`
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`><0
`
`rJ)
`
`CC
`
`Figure 2. The restriction fragments of X174 Hae III DNA from 72 bp
`to 603 bp separated on a CAE chip.
`The separation matrix was 0.75%
`hydroxyethyl cellulose with on-column fluorescent labeling using 1.0 M
`thiazole orange. Excitation was with the 488-nm line of an argon ion
`laser and fluorescence was detected with a photomultiplier tube preceded
`by a bandpass filter centered at 530 nm. A: E=240 V/cm; effective
`separation length (ESL)=3.5 cm. B: E=125 V/cm; ESL=l.0 cm. C: E=250
`V/cm; ESL=l.0 cm.
`
`Historically, DNA fragment sizing has been done using slab gel
`electrophoresis. However, a typical sizing experiment may take from a
`few hours to tens of hours. Recently, our group reduced the time for
`sizing separations down to about 20-30 minutes6 by using CAE.
`Performing DNA fragment sizing on chips has yielded yet another order-
`of-magnitude increase in the speed of separations, reducing the time for
`separations to one to two minutes.14
`
`Figure 2 demonstrates the high-speed separations obtained using CAE
`chips. For all three separations, 10 bp resolution is obtained for the
`271 and 281 bp fragments.
`Figure 2A shows a separation using the
`typical effective separation length (ESL) of 3.5 cm and a field of 240
`
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`V/cm; the time required for the separation is about 100 seconds.
`Figures 23 and 2C show that high-resolution separations can be obtained
`with an ESL as short as 1 cm. With an electric field of 250 V/cm, the
`separation is complete in about 20 seconds. It is amusing to note that
`it took less time for that separation than it has taken you to read this
`paragraph. CAE chips can be used to perform separations with 10 bp
`resolution in under 20 seconds in distances as short as 1 cm!
`
`Figure 3 demonstrates the excellent run-to-run reproducibility of
`DNA separations on chips. Shown are the first three and the last three
`in a series of eight consecutive separations performed on a CAE chip;
`all the fragments in the digest are resolved in each of the runs. The
`small variation in migration times of the fragments (2-3% relative
`standard deviation) is typical for multiple capillary runs.6 We have
`performed as many as 77 separations in a channel with a single HEC
`filling. We are currently working on a chip to perform simultaneous
`separation and detec tion of DNA fragments in 3 2 independent channels.
`
`Allelic sizing is a promising technique for genetic typing and
`forensic identification. Allelic typing of multiple polymorphic STR
`loci is emerging as a technique to test for paternity and personal
`identity.15'16 Figure 4 demonstrates the use of a CAE chip to perform
`rapid allelic typing of the HUMTHO1 locus.
`The sample injected
`consisted of a mixture of DNA from two different samples, one of
`genotype (6/9.3) and the other (7/10) . The peaks corresponding to the
`four alleles are all well-resolved, and the separation took under five
`minutes. Alleles 9.3 and 10 differ in length by a single nucleotide,
`demonstrating that single-base resolution is possible on chips. The use
`of multiplexing17 and a two-color detection system7 would improve the
`throughput of these separations even more. In the future, these chips
`could be used to perform high-speed DNA fingerprinting in a matter of
`seconds or minutes, instead of hours or days.
`
`4. DNA SEQUENCING
`
`The development of high-speed, high-throughput methods for DNA
`sequencing is a crucial challenge of the Human Genome Project.8'9 Our
`group has already developed the technique of capillary array
`electrophoresis which increases the speed of DNA sequencing by an
`order-of-magnitude.35 We have also demonstrated that the speed of
`separations of double-stranded DNA can be increased by an order-of-
`magnitude by performing separations on a CAE chip.14 Can ultra-high-
`speed DNA sequencing also be performed on a chip?
`
`Figure 5 presents the separation of A-terminated M13mp18 DNA
`sequencing fragments on a chip. The separation out to —400 bases takes
`about 10 minutes. The expanded region showing the first 1OO bases
`DNA
`demonstrates that single base resolution has been obtained.
`sequencing with sufficient resolution and signal strength can be
`performed on a CAE chip with another order-of-magnitude improvement in
`the speed!
`
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`C"
`
`en0C
`
`CI
`
`ene
`
`20
`
`10
`
`0I
`
`) tCC
`
`at
`
`Time (seconds)
`
`Figure 3.
`The first three and the last three in a series of 8
`consecutive separations of øXl74 Hae III DNA restriction fragments
`performed on a CAE chip.
`
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`20b
`
`><
`I)
`
`rJ)
`
`00
`
`Time (seconds)
`Figure 4.
`Ultra-high-speed forensic identification of STR alleles.
`The separation medium was 9% T polyacrylamide (0% C) with 7 M urea. The
`fluorescent PCR-amplified alleles were generated with double-FAM-labeled
`primers .'
`
`c1e
`
`><
`
`c,J
`Ct
`
`00
`
`160
`Time (seconds)
`Figure 5. Ultra-high-speed DNA sequencing on a chip. Separation of
`M13mp18 A-sequencing fragments generated with double-FAM-labeled
`primers.18 Bottom: An expanded view of the first l00 bases.
`
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`5. SUMMARY AND PROSPECTS
`
`We have demonstrated the use of CAE chips to perform DNA restriction
`fragment sizing, STR allelic fragment sizing, and DNA sequencing. These
`microfabricated chips can perform separations as much as 1000 times
`faster than with the conventional slab gel methodology. The resolution
`and reproducibility of the separations obtained on our CAE chips are
`co:mparable to that obtained with slab gels and fused silica capillaries.
`
`The prospects for the use of integrated separation devices are
`excellent. A chip could be made to perform ultra-fast genetic typing
`and DNA fingerprinting in minutes or even seconds. CAE chips could be
`used to perform ultra-high-speed, high-throughput DNA sequencing.
`Additionally, chips could be made that combine this high-speed, high-
`resolution separation technology with integrated sample handling. In
`the not too distant future it is conceivable that chips could be used
`for integrated DNA sample preparation, amplification, injection,
`separation, and detection, all in a matter of a few minutes.
`
`6 .
`
`ACKNOWLEDGMENTS
`
`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. Adam Woolley was supported by a
`fellowship by the Fannie and John Hertz Foundation. Microfabrication
`was performed at the University of California, Berkeley Microfabrication
`Laboratory.
`
`7. REFERENCES
`
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`
`2. Dubrow RS. "Capillary Gel Electrophoresis." In: Grossman PD, Colburn
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`
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`, Chakraborty R. "Evaluation of
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