DNA sequencing by capillary electrophoresis (review ... · PDF fileDNA sequencing by capillary...

J. Biochem. Biophys. Methods 41 (1999) 103–119www.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 progressduring 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 sequencingperformance are discussed. Multicapillary array instrumentation is compared with one-capillarysystems. Integrated systems that perform the whole DNA sequencing operation online startingfrom the DNA amplification through base calling and data processing are discussed. 1999Elsevier 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 ofcontemporary biology, and the desire to sequence the complete human genome hasspawned the Human Genome Project [1]. In the past, slab gel electrophoresis dominatedDNA sequencing; now capillary electrophoresis is becoming the dominant technique. Inthe future, hybridization and mass spectrometry may replace electrophoresis forcomparative or diagnostic sequencing [2], but electrophoresis will probably remain themajor tool for de novo sequencing.

The earlier history of the DNA sequencing, including the use of crosslinked gels forcapillary electrophoresis, has been reviewed nicely by Dovichi [3]. Readers interested ina 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 inducedfluorescence; LPA, linear polyacrylamide; M , average molecular mass; PEO, polyethylene oxide; PDMA,a

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.PI I : S0165-022X( 99 )00041-X

´104 V. Dolnık / J. Biochem. Biophys. Methods 41 (1999) 103 –119

Dovichi paper. The current review primarily describes developments in DNA sequencingby 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 thatmake accurate base calling difficult are absolutely essential for efficient reading of DNAsequences. The quality of amplified sequences can be improved by the presence ofbetaine during amplification, which reduces the formation of secondary structure causedby CG-rich regions and thus improves the accuracy of amplification [4].

The use of modular primers consisting of hexamer or pentamer oligonucleotides hasbeen proposed [5,6]. For this, the modules are base-stacked to each other upon annealingto a DNA template. A given combination of modules thus primes a DNA sequencingreaction uniquely. The use of modules can replace synthesis of traditional primers,which is currently the bottleneck in time and cost of primer-walk sequencing. Librariesof short oligonucleotides have been used to prime sequencing of a 3.5-kb stretch of thesingle 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 inSanger DNA sequencing used a solid-phase nanoreactor that is directly coupled to thecapillary electrophoresis instrument [8].

The presence of impurities in DNA samples can have a detrimental effect both on thequality of the current DNA sequencing run and on capillary lifetime. A method ofsolid-phase purification has been developed using primer oligonucleotides containing abiotin moiety at an internal position [9,10]. The primer is used in the enzymaticreactions, and the resulting products are then bound to streptavidin-coated magneticbeads. After washing out the impurities, the DNA sequencing fragments are releasedfrom the beads and analyzed.

Chlorides and other small anions present in the sample do not significantly affect theseparation 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 thecapillary during sampling. Salts are typically removed from the sample by ethanolprecipitation. However, Salas-Solano et al. showed that the content of chloride can stayas high as about 25 mM after the ethanol precipitation [11]. A method using gel filtrationin a spin column format has been developed to remove excessive chlorides [12].Prepared samples should be dissolved in a low conductivity formamide without anysalts, which keeps the transference number of DNA high and thus increases the amountof DNA loaded [13].

Template is another impurity in sequencing samples that can significantly influencethe separation performance of the capillary. The Salas-Solano et al. have found that 0.1mg of template in the sample (one-third the smallest amount recommended in cyclesequencing) decreases the separation efficiency of capillary electrophoresis by 70% [11].

´V. Dolnık / J. Biochem. Biophys. Methods 41 (1999) 103 –119 105

They have developed a method for removing template from the samples using poly(ether5 6sulfone) membranes deactivated with LPA (M 7 ? 10 –10 ) [12].a

3. Sampling

Because of the high viscosity of the majority of sieving matrices, DNA samples aretypically loaded into the capillary by electromigration. Surprisingly, there is not fullagreement on the optimum electric field for this purpose. Some researchers prefer a lowsampling voltage [11,14] or voltage ramping. They speculate that these methodsminimize the generation of Joule heating, thus reducing expansion of the polymersolution out of the capillary and a loss of sample DNA. On the other hand, a fast voltageramp has been found essential for ultrafast analysis of DNA fragments [15].

To minimize peak broadening by sampling, pH-mediated sample concentration has2been proposed [16]. After injection of DNA, a high-pH zone (theoretically of OH ) that

has lower mobility than DNA is applied at the injection site, and this concentrates DNAinto a narrow isotachophoretic zone.

As an alternative approach, silver-coated capillaries have been proposed for directsample injection in DNA sequencing [17]. They are used to sample in the absence ofadded electrolytes.

4. Electrophoretic separation

4.1. Sieving matrix

The first experiments with DNA sequencing by capillary electrophoresis usedcrosslinked polyacrylamide gels. However, the crosslinked-polyacrylamide capillary hada rather limited lifetime [18]. Replaceable sieving matrices of linear polymers wereintroduced [19] with the use of linear polyacrylamide. There were originally somedoubts about performing DNA sequencing in linear polymers because the relaxation

6time of the polymer fiber (M 10 ) is comparable to the residence time of a 100-bp DNAa

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 asignificant decrease in the separation efficiency [21]. Some differences in separationperformance were also observed depending on the horizontal vs. vertical position of thecapillary [19]. These negative effects either have not been confirmed in practice or wereeasily eliminated. With some exceptions [22], replaceable sieving matrices are now usedin DNA sequencing exclusively.

Several polymers have been used successfully in DNA sequencing: linear poly-acrylamide [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 understoodwhy some polymers are better sieving matrices than others. It has been observed that a


larger molecular mass of the polymer means longer readlength but results in poorerseparation of short fragments. Linear polymers are much more suitable for separation oflong DNA sequencing fragments than are branched polymers [40].

Linear polyacrylamide represents the best replaceable sieving polymer today in termsof readlength and speed [41], although formation of a dynamic wall coating by PDMAand PEO, which allows DNA sequencing in bare fused-silica capillaries (see Section 5below), sometimes makes these latter polymers the preferred choice [42]. In our opinion,DNA sequencing performance expressed as the readlength generated per unit time is themore important criterion. The readlength itself depends on a number of factors, namelytemperature, voltage, and the molecular mass of the polymer. It is not clear whypolyacrylamide currently provides the best DNA sequencing data. It may be because ofinherently superior properties of linear polyacrylamide, or it may simply be becauseacrylamide is available in much higher purity than other monomers.

Karger and co-workers showed that linear polyacrylamide can generate DNAsequencing 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 forroutine DNA sequencing (98.5%). The same group developed a method of emulsionpolymerization of linear polyacrylamide [24]. With 2% (w/w) linear acrylamide (Ma

69 ? 10 ), the run-to-run base calling accuracy was (n 5 9): 99.260.1% for the first 800bases and 98.160.3% for the first 900 bases. The published data allow calculation of abatch-to-batch accuracy (n 5 5) of 99.060.3% for the first 800 bases and 98.160.4% forthe first 900 bases.

PEO is another extensively studied sieving polymer. Fung and Yeung used a mixture6 5of two molecular mass populations of PEO, 8 ? 10 and 6 ? 10 , for DNA sequencing by

capillary electrophoresis [31]. It was possible to separate DNA sequencing fragmentsover 1000 bases, but the separation time exceeded 7 h [43]. PEO acts as a dynamic wallcoating in bare fused-silica capillaries [31]. Mixing two populations of polymer, eachwith a narrow but very different range of molecular mass, has become popular because ithelps to tune the separation performance over a broad range of DNA lengths. Theaddition of the shorter sieving polymer improves the separation of short fragmentswithout significantly compromising the separation of the long ones [25,40,43,44].

A rather different strategy was used by Grossman [45] and Madabhushi [34] whoconsidered low-viscosity of the sieving matrix a higher priority than maximumreadlength. Low viscosity of the sieving matrix translates into lower pressure for fillingand rinsing the capillary and thus safer operation, shorter filling times, and so on.However, this means forgoing on long readlengths. With PDMA, maximum readlengthsof about 600 bases are obtained. Nevertheless, PDMA has the advantage of being apowerful agent in reducing electroosmotic flow (EOF). PVP [35] is another lowviscosity sieving matrix that can be used in bare capillaries. It provides a readlength ofapproximately 500 bases.

A promising type of medium composed of polyethylene glycol end-capped withmicelle-forming fluorocarbon tails has been tested by Menchen et al. [36,37]. Theinteractions between fluorocarbon tails leads to the formation of an equilibrium networkwith well defined mesh size. Under shear the network breaks down, which allowsreplacement of the matrix in the capillary between runs.


Fig. 1. Capillary electrophoresis of DNA sequencing fragments generated on M13 mp18 using 2% LPA (Ma6 49 ? 10 ) and 0.5% LPA (M 5 ? 10 ) as sieving polymer. After Ref. [25] with permission.a


The interface between the sieving polymer in the capillary and the free electrolyte inthe reservoirs at the ends of the capillary can be problematic. At this boundary thetransference number of migrating ions is changed, and this results in formation of amigrating concentration boundary [46,47]. Using sieving polymer in the electrodereservoirs at both ends of the separation capillary eliminates this effect.

There have been some efforts to replace the sieving matrix with other separationmedia and/or to use other principles. A promising new concept of nanofabricated arrayshas been tested. In this approach, a series of obstacles is placed in the migration path ofDNA sequencing fragments [48], and the obstacles act to retard the DNA migration.DNA sequencing by capillary electrophoresis in free electrolytes has been proposed byMayer at al. [49]. For this, DNA is labeled with a protein or another monodispersechemical to generate additional friction. The authors predict resolution over 2000 basesin minutes; practical verification is still needed. Another concept of capillary electro-phoresis of DNA in free electrolyte utilizes sub-micron capillaries where the capillaryradius is smaller than the size of the electric double layer [50].

4.2. Denaturants

Compressions are occasionally found during the separation of DNA sequencingfragments, particularly in GC-rich regions. Dovichi et al. found that addition of at least10% formamide improves the denaturing capacity of the sieving matrix and reducescompressions [51]. Rosenblum et al. suggested the addition of 2-pyrrolidinone and atemperature increase (to 608C) to reduce compressions and improve sizing accuracy[52]. Lindberg and Roeraade proposed separating DNA sequencing fragments bycapillary electrophoresis in a non-aqueous medium. In this scenario, N-methylformamideis 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 andreadlength depend on the electric field applied. A high electric field reduces themigration time of DNA molecules. On the other hand, it decreases the size of DNA atwhich migration enters the reptation-with-orientation mode, where all larger DNAmolecules migrate with the same mobility and do not separate.

Inoue et al. applied an electric field step gradient and found that it improved theseparation of DNA sequencing fragments larger than 500 bases [54]. In anotherapproach, the positive effect of very low electric fields on the separation of DNAsequencing fragments was demonstrated by Kim and Yeung [43]. An electric field of 75V/cm allowed separation of 1000 bases, but the electrophoresis required about 7 h. Asimilar 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 DNAsequencing, 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.


In 1992, Slater and Drouin predicted that pulsed-field capillary electrophoresis mightbe a useful tool for increasing the sequencing readlength [57]. Kim and Yeung tested theidea by changing a number of parameters such as waveform, frequency, modulationdepth, and heating [58]. However, they did not see any improvement in separationcompared to the direct current mode. They found that the temperature changes generatedby either pulsed field or ultrasound can have a deleterious effect on the separationefficiency.

4.4. Temperature effect

A study of activation energy clearly showed two different separation modes (Ogstonand reptation) in capillary electrophoresis of DNA sequencing fragments [28]. Experi-ments confirmed that increasing the temperature extends the reptation-without-orienta-tion mode to longer fragments [59,60]. Under fixed run conditions, fragments ofapproximately 600 bases show the best separation efficiency. For these 600-basefragments, 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 andthus can be used for DNA sequencing in bare fused-silica capillaries. This is true forPDMA [34], PEO [31], and PVP [35]. However, the lifetime of bare capillaries islimited because their performance deteriorates with repeated runs, and extensivecapillary rinsing is required between runs. Madabhushi compared electroosmosis (EEO)

29 2 21 21in bare capillaries (m 5 62.9 ? 10 m V s ) filled with solutions of variousEEO

polymers [34]. He found the dynamic coating of PDMA to be the most effective in29 2 21 21reduction of EEO (m 5 2.0 ? 10 m V s ) and polyacrylamide to be leastEEO

29 2 21 21effective (m 5 24.3 ? 10 m V s ).EEO

Both the poor dynamic wall coating ability of the otherwise superb polyacrylamideand a quick deterioration of DNA sequencing performance in bare capillaries cause thatthe permanent wall coating has not become obsolete. The wall coating procedure

´developed by Hjerten [61] was successfully applied to DNA sequencing [33], but thiscoating 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 stablewall 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 acrylamidederivatives, acryloylaminoethoxyethylglucopyranose and acryloyldiethanolamine (bishy-droxyethylacrylamide) [63], and the lifetime of the wall coating exceeded 500 runs (Fig.2). Another wall coating used in DNA sequencing is polyvinylalcohol [64]. The coatinghas a strong interaction with borate buffer creating a negatively charged surface and thusrequiring use of a non-borate buffer for DNA sequencing.


Fig. 2. The quintuplet /quadruplet region in the T-track of M13mp18, electrophoresis in capillaries coated withADEA, run 500. After Ref. [63] with permission.

6. DNA labeling

Laser-induced fluorescence (LIF) detection is the standard detection method in DNAsequencing by capillary electrophoresis. Several labeling strategies have been developed.With some exceptions [65], the DNA primer labeling is done at the 59 end. Recentlyterminator labeling, which is becoming a method of choice has been used [66]. Onelabeling approach uses primers labeled with four dyes (FAM, JOE, ROX and TAMRA),


which have been standard since the beginning of DNA sequencing by capillaryelectrophoresis [67]. These are typically used with fluorescence excited at 488 nm (argonion laser) and 543 nm (helium–neon laser). A four-color confocal fluorescence capillaryarray 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) simplifiesthe detection. Two PCR reactions are started with the primer labeled identically with onedye. The corresponding dideoxynucleotides are used at different concentration ratios, sothat the difference in signal intensity of the second dye relative to the primer dye can beused for base reading [29,69–71].

Fluorescence energy transfer (ET) dyes for labeling DNA primers have beensynthesized by the Mathies and co-workers [72,73]. These primers carry a fluoresceinderivative (FAM) at the 59 end as a common donor and a suitable fluorescein orrhodamine derivative attached to a modified thymidine within the primer sequence as anacceptor. Adjustment of the donor–acceptor spacing through their placement in theprimer sequence allowed tuning the properties and generation of four primers, all havingstrong absorption at a common excitation wavelength (488 nm) and fluorescencemaxima of 525, 555, 580 and 605 nm. The efficiency of energy transfer of these primersrange from 65% to 97%. With argon-ion laser excitation, the fluorescence of the ETprimers is two- to six-fold greater than that of corresponding primers labeled with asingle dye. New ET primers have been synthesized replacing JOE with 5- or 6-carboxyrhodamine-6G as acceptor [74].

A group of new cyanine energy-transfer dyes has been synthesized by the Mathies etal. [75]. These ET dyes are formed by 3(e-carboxypentyl)-39-ethyl-5,59-dimethylox-acarbocyanine dye as a common energy donor and fluorescein or rhodamine derivatives(FAM, R6G, TAMRA and ROX) as acceptor. The intensity of the fluorescence emissionof these ET primers is 1.4- to 24-fold stronger than that of the corresponding primerslabeled with a single fluorophore only. When compared with the corresponding ETprimers with FAM as a donor, the new ET dyes provide 0.8–1.7-fold more fluorescenceemission.

A set of four energy transfer dyes has been synthesized with 5- or 6-carboxyderiva-tives of 49-aminomethylfluorescein as the donor and 4,7-dichloro-substituted rhodaminedyes as acceptors [76,77]. These dyes exhibit narrower emission spectra than theunsubstituted rhodamines. The overall improvement in signal-to-noise is four- tofive-fold.

Three-wavelength detection strategy using four energy-transfer primers has beenapplied to DNA sequencing [78]. The base calling accuracy was comparable to thatusing four-color coding.

Other labeling models have also been investigated. For example, three differentfour-dye systems have been proposed and tested for on-the-fly lifetime detection; twowere excited at 488 nm, one at 514 nm [79]. In another approach, near-infraredfluorescent dyes were synthesized with an isothiocyanate functional group to allow

221attachment to primers [80,81]. The detection limit was found to be 3.4 ? 10 mole ofprimer.

The properties of four classes of fluorescence labels have been studied:dipyrromethene-boron difluoride (BODIPY), ET dyes, conventional fluorescein and


rhodamine dyes [82], and cyanine-dye derivatives [83]. The ET dyes provided highsensitivity but low photostability. The BODIPY dyes showed minimum but nonzeromobility shifts. DNA labeled with certain pairs of cyanine-dye derivatives were found tomigrate 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 DNAsequencing instrumentation to function as a high-throughput system. Array instrumentstypically use about 100 capillaries, however Dovichi has developed instrumentationusing 576 capillaries [3]. Capillary array instruments put special requirements on thedetection system. The first instrument, which was described by the Mathies andco-workers [84,85], used a confocal fluorescence scanner. In another approach, a CCDcamera has been used [3,86–88] to simultaneously record the fluorescence signals fromall capillaries at the rate 0.6 frame per second. To eliminate light scattering, detectionwas performed at a multiple sheath-flow region [89]. Optical waveguides have beenproposed to collect fluorescence in the detection cell [90]. A post-electrophoresiscapillary scanning method has been developed to detect DNA sequencing fragments[91,92].

The confocal fluorescence-scanning method is used in the first commercial system forDNA sequencing by capillary array electrophoresis, the MegaBACE 1000 by MolecularDynamics (Fig. 3) [33,93–95]. The instrument has 96 capillaries [63 cm (40 cmeffective length) 3 75 mm I.D. 3 200 mm O.D.] with the sieving matrix pumped in andout 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) bandpassor 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] withpermission.


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 afrequency 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 andfiltering, is registered by two photomultiplier tubes (PMTs). Temperature control allowsthermostating 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 of200–500 p.s.i. (1.4 MPa–3.4 MPa). Temperature control will be available. Up to eight96-well plates can be housed in the instrument carousel. The migration of DNAfragments will be monitored by a CCD camera, with two spectral channels used forDNA detection.

Three companies producing or developing a capillary electrophoresis DNA sequenceras 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 forDNA sequencing by capillary electrophoresis [100–103]. Capillary electrophoresis on achip brings new potentials into DNA sequencing and after solving some technicalproblems it may become a starting point for an instrumentation of future.

High-throughput DNA sequencing generates large amounts of data. This puts heavyrequirements 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 beperformed automatically in one integrated system. Swerdlow et al. designed anintegrated system where thermal cycling, product purification, in-line loading, andcapillary electrophoresis are coupled in a single instrument [111]. Samples in the loop ofan injection valve are amplified inside an air thermocycler. Impurities are removed fromthe sample by liquid chromatography. The sample is loaded in a continuous flow manneronto a polymer-filled electrophoresis capillary where DNA sequencing fragments areseparated and detected by LIF. During the electrophoretic run, the system is re-conditioned for injection of another sample. Cycle sequencing and electrophoresis of onesample is completed in 90 min.

A similar system has been developed by Tan and Yeung [112]. A dye-labeledterminator cycle-sequencing reaction is performed in a fused-silica capillary. Thesequencing ladder is directly injected into a size-exclusion chromatography column at958C for purification. High temperature prevents the renaturation of DNA fragments.Online injection into the capillary is accomplished at a junction. The system has beenimproved to process eight samples simultaneously (Fig. 4) [108]. The raw data allowbase calling up to 460 bases with an accuracy of 98%. The system is scalable to a96-capillary array.


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 wouldlike to thank Drs. Kate Bechtol and Stevan Jovanovich for discussing the manuscript.

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