J. Biochem. Biophys. Methods 41 (1999) 103–119
www.elsevier.com / locate / jbbm
DNA sequencing by capillary electrophoresis (review)
´ *
Vladislav Dolnık
Molecular Dynamics, 928 East Arques Avenue, Sunnyvale, CA 94086, USA
Abstract
DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress
during the last four years. The effects of sample purification, composition of sieving matrices,
electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing
performance are discussed. Multicapillary array instrumentation is compared with one-capillary
systems. Integrated systems that perform the whole DNA sequencing operation online starting
from the DNA amplification through base calling and data processing are discussed.  1999
Elsevier Science B.V. All rights reserved.
Keywords: DNA sequencing; Capillary electrophoresis; Sieving matrix; Entangled polymer; Wall coating
1. Introduction
Determining the sequence of bases in DNA has become a major challenge of
contemporary biology, and the desire to sequence the complete human genome has
spawned the Human Genome Project [1]. In the past, slab gel electrophoresis dominated
DNA sequencing; now capillary electrophoresis is becoming the dominant technique. In
the future, hybridization and mass spectrometry may replace electrophoresis for
comparative or diagnostic sequencing [2], but electrophoresis will probably remain the
major tool for de novo sequencing.
The earlier history of the DNA sequencing, including the use of crosslinked gels for
capillary electrophoresis, has been reviewed nicely by Dovichi [3]. Readers interested in
a deeper knowledge of that era or in a slightly different point of view are referred to the
Abbreviations: EEO, electroosmosis; ET, energy transfer; HEC, hydroxyethylcellulose; LIF, laser induced
fluorescence; LPA, linear polyacrylamide; Ma , average molecular mass; PEO, polyethylene oxide; PDMA,
polydimethylacrylamide; PVP, polyvinylpyrrolidone; CCD, charge
*Tel.: 1 1-408-7374-810; fax: 1 1-408-7738-343.
´
E-mail address: [email protected] (V. Dolnık)
0165-022X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0165-022X( 99 )00041-X
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Dovichi paper. The current review primarily describes developments in DNA sequencing
by capillary electrophoresis during the last four years.
2. Sample preparation
Sample preparation is a very important part of each DNA sequencing protocol.
Accurate amplification of the studied DNA sequence and removal of impurities that
make accurate base calling difficult are absolutely essential for efficient reading of DNA
sequences. The quality of amplified sequences can be improved by the presence of
betaine during amplification, which reduces the formation of secondary structure caused
by CG-rich regions and thus improves the accuracy of amplification [4].
The use of modular primers consisting of hexamer or pentamer oligonucleotides has
been proposed [5,6]. For this, the modules are base-stacked to each other upon annealing
to a DNA template. A given combination of modules thus primes a DNA sequencing
reaction uniquely. The use of modules can replace synthesis of traditional primers,
which is currently the bottleneck in time and cost of primer-walk sequencing. Libraries
of short oligonucleotides have been used to prime sequencing of a 3.5-kb stretch of the
single stranded M13mp18 template [7].
Reagent expense in general is a major contributor to the overall cost of sequencing.
One approach that has been introduced to minimize the amount of reagents required in
Sanger DNA sequencing used a solid-phase nanoreactor that is directly coupled to the
capillary electrophoresis instrument [8].
The presence of impurities in DNA samples can have a detrimental effect both on the
quality of the current DNA sequencing run and on capillary lifetime. A method of
solid-phase purification has been developed using primer oligonucleotides containing a
biotin moiety at an internal position [9,10]. The primer is used in the enzymatic
reactions, and the resulting products are then bound to streptavidin-coated magnetic
beads. After washing out the impurities, the DNA sequencing fragments are released
from the beads and analyzed.
Chlorides and other small anions present in the sample do not significantly affect the
separation itself, but do result in reduced amounts of the DNA loaded onto the column.
This is because they have higher mobility than DNA and preferentially enter the
capillary during sampling. Salts are typically removed from the sample by ethanol
precipitation. However, Salas-Solano et al. showed that the content of chloride can stay
as high as about 25 mM after the ethanol precipitation [11]. A method using gel filtration
in a spin column format has been developed to remove excessive chlorides [12].
Prepared samples should be dissolved in a low conductivity formamide without any
salts, which keeps the transference number of DNA high and thus increases the amount
of DNA loaded [13].
Template is another impurity in sequencing samples that can significantly influence
the separation performance of the capillary. The Salas-Solano et al. have found that 0.1
mg of template in the sample (one-third the smallest amount recommended in cycle
sequencing) decreases the separation efficiency of capillary electrophoresis by 70% [11].
´ / J. Biochem. Biophys. Methods 41 (1999) 103 – 119
V. Dolnık
105
They have developed a method for removing template from the samples using poly(ether
sulfone) membranes deactivated with LPA (Ma 7 ? 10 5 –10 6 ) [12].
3. Sampling
Because of the high viscosity of the majority of sieving matrices, DNA samples are
typically loaded into the capillary by electromigration. Surprisingly, there is not full
agreement on the optimum electric field for this purpose. Some researchers prefer a low
sampling voltage [11,14] or voltage ramping. They speculate that these methods
minimize the generation of Joule heating, thus reducing expansion of the polymer
solution out of the capillary and a loss of sample DNA. On the other hand, a fast voltage
ramp has been found essential for ultrafast analysis of DNA fragments [15].
To minimize peak broadening by sampling, pH-mediated sample concentration has
been proposed [16]. After injection of DNA, a high-pH zone (theoretically of OH 2) that
has lower mobility than DNA is applied at the injection site, and this concentrates DNA
into a narrow isotachophoretic zone.
As an alternative approach, silver-coated capillaries have been proposed for direct
sample injection in DNA sequencing [17]. They are used to sample in the absence of
added electrolytes.
4. Electrophoretic separation
4.1. Sieving matrix
The first experiments with DNA sequencing by capillary electrophoresis used
crosslinked polyacrylamide gels. However, the crosslinked-polyacrylamide capillary had
a rather limited lifetime [18]. Replaceable sieving matrices of linear polymers were
introduced [19] with the use of linear polyacrylamide. There were originally some
doubts about performing DNA sequencing in linear polymers because the relaxation
time of the polymer fiber (Ma 10 6 ) is comparable to the residence time of a 100-bp DNA
fragment. It was believed that this would prevent long readlengths [20]. In addition,
coiling or uncoiling of capillaries filled with a solution of linear polymer causes a
significant decrease in the separation efficiency [21]. Some differences in separation
performance were also observed depending on the horizontal vs. vertical position of the
capillary [19]. These negative effects either have not been confirmed in practice or were
easily eliminated. With some exceptions [22], replaceable sieving matrices are now used
in DNA sequencing exclusively.
Several polymers have been used successfully in DNA sequencing: linear polyacrylamide [19,23–29], polyethylene oxide (PEO) [30–32], hydroxyethylcellulose
(HEC) [33], polydimethylacrylamide (PDMA) [34], polyvinylpyrrolidone (PVP) [35],
polyethylene glycol with a fluorocarbon tail [36,37], polyacryloylaminopropanol [38],
and a copolymer of acrylamide and allylglucopyranose [39]. It is not well understood
why some polymers are better sieving matrices than others. It has been observed that a
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larger molecular mass of the polymer means longer readlength but results in poorer
separation of short fragments. Linear polymers are much more suitable for separation of
long DNA sequencing fragments than are branched polymers [40].
Linear polyacrylamide represents the best replaceable sieving polymer today in terms
of readlength and speed [41], although formation of a dynamic wall coating by PDMA
and PEO, which allows DNA sequencing in bare fused-silica capillaries (see Section 5
below), sometimes makes these latter polymers the preferred choice [42]. In our opinion,
DNA sequencing performance expressed as the readlength generated per unit time is the
more important criterion. The readlength itself depends on a number of factors, namely
temperature, voltage, and the molecular mass of the polymer. It is not clear why
polyacrylamide currently provides the best DNA sequencing data. It may be because of
inherently superior properties of linear polyacrylamide, or it may simply be because
acrylamide is available in much higher purity than other monomers.
Karger and co-workers showed that linear polyacrylamide can generate DNA
sequencing readlengths beyond 1000 bases with an accuracy of almost 97% in 80 min
(Fig. 1) [23,25]. This accuracy is, however, below the value generally accepted for
routine DNA sequencing (98.5%). The same group developed a method of emulsion
polymerization of linear polyacrylamide [24]. With 2% (w / w) linear acrylamide (Ma
9 ? 10 6 ), the run-to-run base calling accuracy was (n 5 9): 99.260.1% for the first 800
bases and 98.160.3% for the first 900 bases. The published data allow calculation of a
batch-to-batch accuracy (n 5 5) of 99.060.3% for the first 800 bases and 98.160.4% for
the first 900 bases.
PEO is another extensively studied sieving polymer. Fung and Yeung used a mixture
of two molecular mass populations of PEO, 8 ? 10 6 and 6 ? 10 5 , for DNA sequencing by
capillary electrophoresis [31]. It was possible to separate DNA sequencing fragments
over 1000 bases, but the separation time exceeded 7 h [43]. PEO acts as a dynamic wall
coating in bare fused-silica capillaries [31]. Mixing two populations of polymer, each
with a narrow but very different range of molecular mass, has become popular because it
helps to tune the separation performance over a broad range of DNA lengths. The
addition of the shorter sieving polymer improves the separation of short fragments
without significantly compromising the separation of the long ones [25,40,43,44].
A rather different strategy was used by Grossman [45] and Madabhushi [34] who
considered low-viscosity of the sieving matrix a higher priority than maximum
readlength. Low viscosity of the sieving matrix translates into lower pressure for filling
and rinsing the capillary and thus safer operation, shorter filling times, and so on.
However, this means forgoing on long readlengths. With PDMA, maximum readlengths
of about 600 bases are obtained. Nevertheless, PDMA has the advantage of being a
powerful agent in reducing electroosmotic flow (EOF). PVP [35] is another low
viscosity sieving matrix that can be used in bare capillaries. It provides a readlength of
approximately 500 bases.
A promising type of medium composed of polyethylene glycol end-capped with
micelle-forming fluorocarbon tails has been tested by Menchen et al. [36,37]. The
interactions between fluorocarbon tails leads to the formation of an equilibrium network
with well defined mesh size. Under shear the network breaks down, which allows
replacement of the matrix in the capillary between runs.
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Fig. 1. Capillary electrophoresis of DNA sequencing fragments generated on M13 mp18 using 2% LPA (Ma
9 ? 10 6 ) and 0.5% LPA (Ma 5 ? 10 4 ) as sieving polymer. After Ref. [25] with permission.
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The interface between the sieving polymer in the capillary and the free electrolyte in
the reservoirs at the ends of the capillary can be problematic. At this boundary the
transference number of migrating ions is changed, and this results in formation of a
migrating concentration boundary [46,47]. Using sieving polymer in the electrode
reservoirs at both ends of the separation capillary eliminates this effect.
There have been some efforts to replace the sieving matrix with other separation
media and / or to use other principles. A promising new concept of nanofabricated arrays
has been tested. In this approach, a series of obstacles is placed in the migration path of
DNA sequencing fragments [48], and the obstacles act to retard the DNA migration.
DNA sequencing by capillary electrophoresis in free electrolytes has been proposed by
Mayer at al. [49]. For this, DNA is labeled with a protein or another monodisperse
chemical to generate additional friction. The authors predict resolution over 2000 bases
in minutes; practical verification is still needed. Another concept of capillary electrophoresis of DNA in free electrolyte utilizes sub-micron capillaries where the capillary
radius is smaller than the size of the electric double layer [50].
4.2. Denaturants
Compressions are occasionally found during the separation of DNA sequencing
fragments, particularly in GC-rich regions. Dovichi et al. found that addition of at least
10% formamide improves the denaturing capacity of the sieving matrix and reduces
compressions [51]. Rosenblum et al. suggested the addition of 2-pyrrolidinone and a
temperature increase (to 608C) to reduce compressions and improve sizing accuracy
[52]. Lindberg and Roeraade proposed separating DNA sequencing fragments by
capillary electrophoresis in a non-aqueous medium. In this scenario, N-methylformamide
is simultaneously a solvent and denaturant and PDMA serves as a sieving polymer [53].
4.3. Effect of electric field strength
In agreement with the theory of polyelectrolyte migration, the separation and
readlength depend on the electric field applied. A high electric field reduces the
migration time of DNA molecules. On the other hand, it decreases the size of DNA at
which migration enters the reptation-with-orientation mode, where all larger DNA
molecules migrate with the same mobility and do not separate.
Inoue et al. applied an electric field step gradient and found that it improved the
separation of DNA sequencing fragments larger than 500 bases [54]. In another
approach, the positive effect of very low electric fields on the separation of DNA
sequencing fragments was demonstrated by Kim and Yeung [43]. An electric field of 75
V/ cm allowed separation of 1000 bases, but the electrophoresis required about 7 h. A
similar effect has been observed by Manabe et al. [55] and by Nishikawa and Kambara
[56]. The latter group needed 44 h to resolve 800 bases. In contrast, in ultrafast DNA
sequencing, an electric field strength of 600 V/ cm produced an analysis time of 3–4 min
[15]. However, the readlength was not much above 300 bases.
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In 1992, Slater and Drouin predicted that pulsed-field capillary electrophoresis might
be a useful tool for increasing the sequencing readlength [57]. Kim and Yeung tested the
idea by changing a number of parameters such as waveform, frequency, modulation
depth, and heating [58]. However, they did not see any improvement in separation
compared to the direct current mode. They found that the temperature changes generated
by either pulsed field or ultrasound can have a deleterious effect on the separation
efficiency.
4.4. Temperature effect
A study of activation energy clearly showed two different separation modes (Ogston
and reptation) in capillary electrophoresis of DNA sequencing fragments [28]. Experiments confirmed that increasing the temperature extends the reptation-without-orientation mode to longer fragments [59,60]. Under fixed run conditions, fragments of
approximately 600 bases show the best separation efficiency. For these 600-base
fragments, the maximum separation efficiency was found at a temperature of 608C [11].
5. Wall coating
It has been shown that some sieving polymers can also act as a dynamic coating and
thus can be used for DNA sequencing in bare fused-silica capillaries. This is true for
PDMA [34], PEO [31], and PVP [35]. However, the lifetime of bare capillaries is
limited because their performance deteriorates with repeated runs, and extensive
capillary rinsing is required between runs. Madabhushi compared electroosmosis (EEO)
in bare capillaries ( mEEO 5 62.9 ? 10 29 m 2 V 21 s 21 ) filled with solutions of various
polymers [34]. He found the dynamic coating of PDMA to be the most effective in
reduction of EEO ( mEEO 5 2.0 ? 10 29 m 2 V 21 s 21 ) and polyacrylamide to be least
effective ( mEEO 5 24.3 ? 10 29 m 2 V 21 s 21 ).
Both the poor dynamic wall coating ability of the otherwise superb polyacrylamide
and a quick deterioration of DNA sequencing performance in bare capillaries cause that
the permanent wall coating has not become obsolete. The wall coating procedure
´ [61] was successfully applied to DNA sequencing [33], but this
developed by Hjerten
coating suffers from a limited lifetime caused by hydrolysis of the –Si–O–Si– bond.
The procedure utilizing Grignard chemistry used by Cobb et al. provides a more stable
wall coating [62]. It was further modified to allow large-scale production [14]. However,
the hydrolysis of polyacrylamide limited the lifetime of the capillary to about 110 runs
[14]. Recently, acrylamide was replaced in the procedure with more stable acrylamide
derivatives, acryloylaminoethoxyethylglucopyranose and acryloyldiethanolamine (bishydroxyethylacrylamide) [63], and the lifetime of the wall coating exceeded 500 runs (Fig.
2). Another wall coating used in DNA sequencing is polyvinylalcohol [64]. The coating
has a strong interaction with borate buffer creating a negatively charged surface and thus
requiring use of a non-borate buffer for DNA sequencing.
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Fig. 2. The quintuplet / quadruplet region in the T-track of M13mp18, electrophoresis in capillaries coated with
ADEA, run 500. After Ref. [63] with permission.
6. DNA labeling
Laser-induced fluorescence (LIF) detection is the standard detection method in DNA
sequencing by capillary electrophoresis. Several labeling strategies have been developed.
With some exceptions [65], the DNA primer labeling is done at the 59 end. Recently
terminator labeling, which is becoming a method of choice has been used [66]. One
labeling approach uses primers labeled with four dyes (FAM, JOE, ROX and TAMRA),
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which have been standard since the beginning of DNA sequencing by capillary
electrophoresis [67]. These are typically used with fluorescence excited at 488 nm (argon
ion laser) and 543 nm (helium–neon laser). A four-color confocal fluorescence capillary
array scanner that uses excitation at a single laser wavelength has also been constructed
[68]. In another approach, labeling with only two dyes (FAM and TAMRA) simplifies
the detection. Two PCR reactions are started with the primer labeled identically with one
dye. The corresponding dideoxynucleotides are used at different concentration ratios, so
that the difference in signal intensity of the second dye relative to the primer dye can be
used for base reading [29,69–71].
Fluorescence energy transfer (ET) dyes for labeling DNA primers have been
synthesized by the Mathies and co-workers [72,73]. These primers carry a fluorescein
derivative (FAM) at the 59 end as a common donor and a suitable fluorescein or
rhodamine derivative attached to a modified thymidine within the primer sequence as an
acceptor. Adjustment of the donor–acceptor spacing through their placement in the
primer sequence allowed tuning the properties and generation of four primers, all having
strong absorption at a common excitation wavelength (488 nm) and fluorescence
maxima of 525, 555, 580 and 605 nm. The efficiency of energy transfer of these primers
range from 65% to 97%. With argon-ion laser excitation, the fluorescence of the ET
primers is two- to six-fold greater than that of corresponding primers labeled with a
single dye. New ET primers have been synthesized replacing JOE with 5- or 6carboxyrhodamine-6G as acceptor [74].
A group of new cyanine energy-transfer dyes has been synthesized by the Mathies et
al. [75]. These ET dyes are formed by 3(e-carboxypentyl)-39-ethyl-5,59-dimethyloxacarbocyanine dye as a common energy donor and fluorescein or rhodamine derivatives
(FAM, R6G, TAMRA and ROX) as acceptor. The intensity of the fluorescence emission
of these ET primers is 1.4- to 24-fold stronger than that of the corresponding primers
labeled with a single fluorophore only. When compared with the corresponding ET
primers with FAM as a donor, the new ET dyes provide 0.8–1.7-fold more fluorescence
emission.
A set of four energy transfer dyes has been synthesized with 5- or 6-carboxyderivatives of 49-aminomethylfluorescein as the donor and 4,7-dichloro-substituted rhodamine
dyes as acceptors [76,77]. These dyes exhibit narrower emission spectra than the
unsubstituted rhodamines. The overall improvement in signal-to-noise is four- to
five-fold.
Three-wavelength detection strategy using four energy-transfer primers has been
applied to DNA sequencing [78]. The base calling accuracy was comparable to that
using four-color coding.
Other labeling models have also been investigated. For example, three different
four-dye systems have been proposed and tested for on-the-fly lifetime detection; two
were excited at 488 nm, one at 514 nm [79]. In another approach, near-infrared
fluorescent dyes were synthesized with an isothiocyanate functional group to allow
attachment to primers [80,81]. The detection limit was found to be 3.4 ? 10 221 mole of
primer.
The properties of four classes of fluorescence labels have been studied:
dipyrromethene-boron difluoride (BODIPY), ET dyes, conventional fluorescein and
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rhodamine dyes [82], and cyanine-dye derivatives [83]. The ET dyes provided high
sensitivity but low photostability. The BODIPY dyes showed minimum but nonzero
mobility shifts. DNA labeled with certain pairs of cyanine-dye derivatives were found to
migrate with identical mobilities over a broad range of DNA lengths.
7. Single capillary vs. capillary array
The use of capillary arrays instead of single capillaries allows capillary DNA
sequencing instrumentation to function as a high-throughput system. Array instruments
typically use about 100 capillaries, however Dovichi has developed instrumentation
using 576 capillaries [3]. Capillary array instruments put special requirements on the
detection system. The first instrument, which was described by the Mathies and
co-workers [84,85], used a confocal fluorescence scanner. In another approach, a CCD
camera has been used [3,86–88] to simultaneously record the fluorescence signals from
all capillaries at the rate 0.6 frame per second. To eliminate light scattering, detection
was performed at a multiple sheath-flow region [89]. Optical waveguides have been
proposed to collect fluorescence in the detection cell [90]. A post-electrophoresis
capillary scanning method has been developed to detect DNA sequencing fragments
[91,92].
The confocal fluorescence-scanning method is used in the first commercial system for
DNA sequencing by capillary array electrophoresis, the MegaBACE 1000 by Molecular
Dynamics (Fig. 3) [33,93–95]. The instrument has 96 capillaries [63 cm (40 cm
effective length) 3 75 mm I.D. 3 200 mm O.D.] with the sieving matrix pumped in and
out of the capillaries by high-pressure nitrogen (1000 p.s.i., i.e., 6.9 MPa). The confocal
Fig. 3. Scheme of instrumentation for capillary array electrophoresis. Components: (1) primary beamsplitter,
(2) laser filter, (3) achromat lens, (4) confocal spatial filter, (5) secondary beamsplitter, (6) and (7) bandpass
or longpass filter, (8) and (9) photomultiplier tube (PMT), (10) objective mounted to translation stage, (11)
cathode manifold, (12) capillary detection window mount, (13) anode pressure manifold. After Ref. [94] with
permission.
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113
scanning LIF detection allows four-color chemistry. An argon laser beam (488 nm)
passes through a microscope objective (mounted to a translation stage moving at a
frequency of 1.5 Hz) that focuses the excitation light on the plane of the capillary array.
The fluorescence emission is collected by the same objective and, after splitting and
filtering, is registered by two photomultiplier tubes (PMTs). Temperature control allows
thermostating up to 448C.
Another instrument, SCE9600 by Spectrumedix [96,97], which may be available soon,
reportedly will have 96 capillaries in which the matrix is replaced by pressure of
200–500 p.s.i. (1.4 MPa–3.4 MPa). Temperature control will be available. Up to eight
96-well plates can be housed in the instrument carousel. The migration of DNA
fragments will be monitored by a CCD camera, with two spectral channels used for
DNA detection.
Three companies producing or developing a capillary electrophoresis DNA sequencer
as a product, i.e., Molecular Dynamics [93], Spectrumedix [98], and PE Biosystems [99]
will be participating in the DNA sequencing session and the panel discussion at HPCE
‘99. Unfortunately, this will occur after the deadline of the manuscript for this paper.
Substantial effort is being directed toward microfabrication of a device on a chip for
DNA sequencing by capillary electrophoresis [100–103]. Capillary electrophoresis on a
chip brings new potentials into DNA sequencing and after solving some technical
problems it may become a starting point for an instrumentation of future.
High-throughput DNA sequencing generates large amounts of data. This puts heavy
requirements on software for data handling and data storage [104–110].
8. Integration online
It would be a significant advantage if all steps of DNA sequencing analysis could be
performed automatically in one integrated system. Swerdlow et al. designed an
integrated system where thermal cycling, product purification, in-line loading, and
capillary electrophoresis are coupled in a single instrument [111]. Samples in the loop of
an injection valve are amplified inside an air thermocycler. Impurities are removed from
the sample by liquid chromatography. The sample is loaded in a continuous flow manner
onto a polymer-filled electrophoresis capillary where DNA sequencing fragments are
separated and detected by LIF. During the electrophoretic run, the system is reconditioned for injection of another sample. Cycle sequencing and electrophoresis of one
sample is completed in 90 min.
A similar system has been developed by Tan and Yeung [112]. A dye-labeled
terminator cycle-sequencing reaction is performed in a fused-silica capillary. The
sequencing ladder is directly injected into a size-exclusion chromatography column at
958C for purification. High temperature prevents the renaturation of DNA fragments.
Online injection into the capillary is accomplished at a junction. The system has been
improved to process eight samples simultaneously (Fig. 4) [108]. The raw data allow
base calling up to 460 bases with an accuracy of 98%. The system is scalable to a
96-capillary array.
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Fig. 4. Scheme of an integrated DNA sequencing instrumentation on-line with eight capillaries. After Ref.
[113] with permission.
Acknowledgements
The manuscript was supported by NIH grant 2R44 HG01563-02. The author would
like to thank Drs. Kate Bechtol and Stevan Jovanovich for discussing the manuscript.
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