Methods in Molecular Biology TM VOLUME 162 Capillary Electrophoresis of Nucleic Acids Volume I Introduction to the Capillary Electrophoresis of Nucleic Acids Edited by Keith R Mitchelson Jing Cheng HUMANA PRESS CE for DNA Polymorphism Analysis Overview The Application of Capillary Electrophoresis for DNA Polymorphism Analysis Keith R Mitchelson Introduction The development of capillary electrophoresis (CE) technology has been rapid over the past three years for application to the analytical separation in a variety of biopolymers such as proteins, polysaccharides, and DNA (1–3) CE offers high throughput and high resolution, automatic operation and on-line detection with automatic data acquisition, and this has stimulated its particular application to the analysis of DNA mutations for genetic analysis, and medical diagnosis These advantages have also provided the impetus to the recent miniaturization of CE equipment to silicon-chip based devices (3–12), which provide all of the above facilities, as well as a significant improvement in the speed and degree of automation of analysis Significant developments of other miniaturized electro-separation devices including molecular electrophoresis sieves and dielectric trapping using microelectrodes (13,14) have been described, which may be able to be integrated with CE to create micro-analytical or preparative devices This chapter reviews the development of mutation-detection assays for use with CE CE instrumentation (see Fig 1) consists of two electrolyte chambers linked by a thin capillary, typically of 50–100 µm id The temperature gradients and distortions that can affect the resolution of bands in conventional gels are virtually absent in CE as the microcapillary facilitates rapid heat dissipation, despite the application of large electric fields Data on fractionated molecules is acquired automatically by an on-line detector positioned close to the outlet of the capillary DNA may be detected using the natural UV absorption, although its low sensitivity may limit the detection of samples at low DNA concentrations or low-abundance molecules Laser-induced fluorescence (LIF) (15) provides extremely high sensitivity (approx 100 times UV absorption) through induced detection of additives attached to the DNA, and greatly extends the lower limit of From: Methods in Molecular Biology, Vol 162: Capillary Electrophoresis of Nucleic Acids, Vol 1: Introduction to the Capillary Electrophoresis of Nucleic Acids Edited by: K R Mitchelson and J Cheng © Humana Press Inc., Totowa, NJ Mitchelson Fig Diagrammatic representation of a capillary electrophoresis apparatus The analyte is passaged under an electric field through sieving matrix held within a fine capillary column The passage of the analyte past a window is detected using a photometric device The high surface to volume ratio of the capillary aids dissipation of Joule heat Reprinted from Mitchelson, K R., Cheng, J and Kricka, L J (1997) Use of capillary electrophoresis for point mutation screening Trends in BioTechnology 15, 448–458 Copyright (1997), with the permission of Elsevier Science concentration at which DNA may be detected New instrument designs for more efficient laser excitation and signal detection have been described by Yeung (16), in which the laser light propagates through an array of immersed square capillaries, without undergoing a serious reduction in power The excitation scheme can be potentially scaled up to hundreds of capillaries to achieve high speed and extremely high throughput Sieving Media, Electrolytes As important as the development of new protocols and applications, new electrophoretic sieving-media show potential for high resolution of DNA molecules based on shape (17) and length (18–20) Novel sieving media, with copolymers between acrylamide and β-D-glucopyranoside and glucose producing a medium with highresolving capacity and low viscosity (18) improve media exchange and handling Similarly, media comprising block copolymers (21–23) allow high-resolution sieving matrices to be formed in capillary from low viscosity precursors New electrophoretic procedures have also been developed that allow better separation of short DNA mol- CE for DNA Polymorphism Analysis ecules (500 bp) An electrophoresis protocol such a “temperature programmed electrophoresis” (26,27), which produces temperature microenvironments in the capillary, allows for efficient separation of heteroduplex DNA molecules based on DNA conformation Another procedure, “variable-field electrophoresis” (20) in which both electric field (and temperature) may be modulated during a run provides for improved separation of singlestrand molecules during high-resolution DNA sequencing Stepwise electric field gradients are useful for both sizing experiments (17) and for DNA sequencing of longer fragment (20) Combinations of novel media, buffers, and electrophoresis procedures will continue to provide new paradigms for resolution of DNA and oligonucleotides An example is the new type of grafted copolymer medium, poly(N-isopropylacrylamide)-g-poly(ethylene oxide) (PNI-PAM-g-PEO) solution, which self-coats the capillary tubing (28) A φX174/HaeIII digest could be separated within 24 s using an 8% w/v PNIPAM-g-PEO solution in a 1.5-cm long column with a field voltage of 2400 V DNA Mutation Detection The detection of DNA mutations and natural variation has become central to the characterisation and diagnosis of human genetic diseases and is a core to many aspects of molecular biology and medicine/genetics Several recent reviews provide detailed descriptions of CE applications developed for the detection of point mutations (3,4,26,29,30) Most methods of mutation detection (see Table 1) can be classified into two general categories: (1) methods to detect known mutations and (2) methods to detect unknown mutations Known mutations in target loci are detected by employing various techniques including DNA sequencing (15,20), DNA mini-sequencing and allele-specific amplification (ASA) (31–33), selective primer sequencing (34–36), amplification-refractory-mutation assay (ARMS) (37), and the ligase chain reaction (LCR) (38,39) Other methods that examine changes in defined DNA regions such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (40,41) and short tandem repeat (STR) length polymorphism (42–47) are also used to identify mutations The methods for detecting unknown mutations in DNA fragments include DNA sequencing, single-strand-conformation polymorphism (SSCP) (48–51), heteroduplexpolymorphism assay (HPA) (52,53), constant denaturant (54,55) and denaturing gradient gel electrophoresis (DGGE) (29,56) and chemical or enzymatic cleavage of mismatches (CMC or EMC) (57–64) Frequently, combinations of several complementary techniques are employed to characterize an unknown mutation 3.1 Polymorphism Detection by DNA Sequencing, Sizing, and Quantification CE can size-fractionate DNA fragments up to several kilobases in less than 20 It has been successfully adapted to standard analytical techniques, in which multiple Table Mitchelson Abbreviations and Mutation Detection Methods Summary guide to abbreviations ASA, allele-specific amplification AFLP, amplified fragment length polymorphism ARMS, amplification refractory mutation system ACE, array capillary electrophoresis Block co-polymer sieving media CAGE, capillary affinity gel-electrophoresis CAE, capillary array electrophoresis Capillary coating materials CDGE, constant denaturant capillary gel electrophoresis CGE, non-denaturing capillary gel electrophoresis CEMSA, capillary electrophoresis mobility shift assay CE/MS, capillary electrophoresis/ mass spectrography Chip capillary electrophoresis CMC, chemical mismatch cleavage Collection of capillary electrophoresis sample fractions Co-polymer sieving media CZE, capillary zone electrophoresis DGGE, denaturing gradient gel electrophoresis DD RT-PCR, differential display reverse-transcriptasepolymerase chain reaction DNA sequencing by capillary electrophoresis DOP-PCR, Degenerate oligonucleotide primedpolymerase chain reaction Electric field strength EMC, enzymatic mismatch cleavage ESCE, entangled-solution capillary electrophoresis HPA, heteroduplex DNA polymorphism assay Integrated micro-analytical device Isothermal DNA amplification LCR, ligase chain reaction LIF, laser-induced fluorescence LIFP, laser-induced fluorescence polarization Microfabrication inside the capillary Minisequencing/ primer extension MIDAS, mismatch cleavage DNA analysis system PCR-CE, automated polymerase chain reaction and capillary electrophoresis assay PF-CE, pulsed-field capillary electrophoresis Pyrosequencing Q RT-PCR, quantitative reverse-transcriptase-polymerase chain reaction Reference to conventional mutationdetection methods 31–33 34,36 Reference to analysis using capillary electrophoresis 29 37 8,16,76,106 21,22,28 97–100,105 43–46,76,80,89 19 54,55 24–27,29,37 101–104 110–119 3–13,105–108 62 125–127 18,23,81,85 24–27,29,37,71 29 1,2 67–69,74 15,20,76–82 97 63,64 1,2,29 57–59 90–92 31 1,2 124 67 10 1,2,20,26 58,60,61 21–24,39,46,53,85 52,53 3–6,105,107,108 93 38,39 15,16,38 100 128 32,33 60,61 6,10,96 1,2,81,83–87 66–73 CE for DNA Polymorphism Analysis Table (continued) Summary guide to abbreviations Quantification of genomic alleles Radial capillary array electrophoresis microplate Selective primer amplification analysis Sieving media SSCP, single-stranded DNA conformation polymorphism STR, short tandem (microsatellite) repeat TPCE, temperature-programmed capillary electrophoresis Ultra-fast capillary electrophoresis Reference to conventional mutationdetection methods 34,35 30 Reference to analysis using capillary electrophoresis 37,71 127 34–36 17–23,83–85 48–51 12,43–46 26,27,29,48,49 88,89 DNA species are size fractionated and parallel analyses are compared for differences in fragment profiles Such parallel analyses include PCR-RFLP of gene loci (40,41), for characteristic repeated DNA length polymorphism’s such as the bacterial terminal RFLP (T-RFLP) of ribosomal genes (47), RAPD polymorphisms (65), amplified fragment length-polymorphism (AFLP) genetic markers (34,36) and for the analysis of simple tandem repeats (STR) (43–46) With pressurized or careful electrokinetic loading of samples, CE can be used for quantification of relative amounts of an individual DNA species within a mixture of DNAs (37,66–71) The direct estimation of the concentration of analytes during CE can be used for the quantification of PCR fragments and applied to the estimation of allele frequency in genome analysis (37,71) 3.1.1 DNA Sequencing by CE DNA sequencing by CE is increasingly reported (1,15,20,77–80) to offer high reproducibility and greatly increased speed compared to planar gels, with elimination of problems associated with electrophoretic distortion and lane tracking (75,76) Array capillary sequencing allows for simple handling of multiple sample changeovers and very high throughput with sequence reads of more than 1000 bases within 80 using ACE (76,77,80) In addition, several technical advances such as, thermal ramping programs (20), pulsed-field electrophoretic separation (81) have resulted in improved base-calling and higher resolutions particularly for long DNA fragments, resulting in cost saving through longer sequence reads (80) An on-column method of sample concentration for capillary-based DNA sequencing was achieved simply by electrokinetic injection of hydroxide ions (82) Field focusing occurs upon neutralization of the cationic Tris buffer, resulting in a zone of lower conductivity Even unpurified products of dye-primer sequencing reactions are concentrated at the front of this low-conductivity zone allowing sample injection times as long as 360 s at 50 V/cm Both resolution and signal strength are excellent relative to highly purified samples and a resolution of at least 0.5 can be generated for fragments up to 650 nt long Mitchelson 3.1.2 Pulsed-Field CE Pulsed-field gel electrophoresis formats have found wide application for the improved separation of large (20–100 kb) and very large (several Mb) DNA molecules, however separations are slow because of the low field strength and low mobility of large molecules in solid gels In a CE format, both entangled solution sieving media (83–85) and field inversion capillary electrophoresis (FICE) (86) have been applied for separation of both large and very large DNA molecules with an improvement in speed by 1–2 orders of magnitude (1,2,87) FICE has also been found to improve the resolution of ssDNA fragments for DNA sequencing, particularly improving resolution of longer fragments and increasing the length of sequence read (20,81) Pulsed-field CE methods would be suited to a microdevice format where very substantial gains in speed of separation would also be realized 3.1.3 Mini-Sequencing and Single-Nucleotide Primer Extension Mini-sequencing is an assay in which a probe is extended by single labeled dideoxy terminator nucleotide if the correct allele is available as template, and incorporation of specific labeled nucleotides can simultaneously identify several SNP alleles at a locus (31) The application of capillary electrophoresis-laser-induced fluorescence (CE-LIF) for detecting single-nucleotide primer extension (SNuPE) products detected three different point mutations in human mitochondrial DNA (32) SNuPE analysis using CE-LIF provides high speed and has the potential for multiplexing with the provision of differentially labeled primers 3.1.4 Selective Primer Amplification and Sequencing Kambara and colleagues (34–36) have developed a selective polymerase chain reaction (PCR) using two-base anchored primers to improve the amplification specificity and eliminate base-mispair amplification This selectivity has been applied to the improvement of genetic marker technology, specifically to the AFLP assay with high fidelity, which when coupled with CE analysis, provides for rapid genotyping and for identification of linked gene markers This selective amplification primer approach can also be used for amplifying one fragment from a DNA fragment mixture (34,35) which may then be classified by CE analysis according to its terminal-base sequences and its length Fragments produce characteristic electropherograms, which may be used to select PCR reaction primers for any fragment in a digestion mixture Comparison of the electropherograms of two different DNA strands allows selective amplification and specific sequencing of several kilobases of DNA without subcloning, which dramatically simplifies DNA fragment analysis 3.2 Refractory Amplification Systems 3.2.1 Amplification Refractory Mutation System ARMS is a PCR-based assay for mutations at known loci in which PCR primers fully complementary to particular alleles amplify a defined product, whereas other alleles are refractory to amplification Rapid diagnosis of the classic form of human 21-hydroxylase deficiency is achieved by the simultaneous detection of common point CE for DNA Polymorphism Analysis mutations in the P450c21 B gene by nested PCR-ARMS in conjunction with capillary zone electrophoresis (CZE) in sieving liquid polymers (37) The common mutations in the CFTR gene are detected using ARMS in conjunction with entangled solution capillary electrophoresis (ESCE) In the first PCR, genes are selectively amplified, then in the nested reaction ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE and detected by the fluorescent dye SYBR Green I using LIF detection The PCR reaction products could be separated without desalting of samples using the CZE and detected with LIF, without sample preconcentration (37) 3.2.2 Ligase Chain Reaction Ligase chain reaction (LCR) is a thermocycler-based assay for known mutations in which oligonucleotide primers fully complementary at loci to particular alleles amplify a defined ligation product, whereas on other alleles, primers not align fully and are thus refractory to cyclic-ligation Since the dsDNA ligation products are short (typically ~ 50–100 bp) and can be rapidly separated from unincorporated ligation primers (20–25 bp) the LCR mutation assay is suited to very rapid CE separation techniques Indeed, CE-LIF using short capillary columns (7.5-cm effective length) and fields of 400 V/cm has been used to simultaneously detect three point mutations in human mitochondrial DNA resulting in Leber’s hereditary optic neuropathy (LHON) with high speed (38) CE-LIF has also been utilized for the rapid separation and highly sensitivity quantitative detection of