Effective Capillary Electrophoresis-Based Heteroduplex Analysis

Anal. Chem. 2000, 72, 5483-5492

Effective Capillary Electrophoresis-Based Heteroduplex Analysis through Optimization of Surface Coating and Polymer Networks Huijun Tian,† Lawrence C. Brody,‡ David Mao,§ and James P. Landers*,|,⊥

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, J&W Scientific, Folsom, California 95630, Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, and Department of Pathology, University of Virginia Medical Center, Charlottesville, Virginia 22908

The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C>G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (γ-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylpyrrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT-PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FCcoated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT-PVP-coated capillary and HEC as the polymer network. The detection of DNA mutations has become an integral part of understanding and diagnosing a variety of diseases, including cancer.1-3 Although DNA sequencing is the accepted “gold * Corresponding author: (phone) 804-243-8658; (fax) 412-243-8852 (secure); (e-mail) [email protected]. † University of Pittsburgh. ‡ National Institutes of Health. § J&W Scientific. | Department of Chemistry, University of Virginia. ⊥ Department of Pathology, University of Virginia Medical Center. (1) Gerrard, B.; Dean, M. Mutation Detection A Practical Approach; Oxford University Press Inc.: New York, 1998. 10.1021/ac0004916 CCC: $19.00 Published on Web 10/04/2000

© 2000 American Chemical Society

standard” for identifying specific nucleotide variations, the high cost of screening samples for mutations combined with the difficulty of detecting heterozygotes by DNA sequencing remains an issue. Accordingly, a variety of methods, including heteroduplex analysis (HDA), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), denaturing highperformance liquid chromatography (DHPLC), primer extension, allele-specific amplification, allele-specific oligonucleotide hybridization, and oligonucleotide ligation, have been developed for DNA mutation detection.1,4-10 Among the above methods, HDA is one of the most widely used methods for detecting unknown mutations.1,4 Traditionally, 32P-labeled deoxynucleoside triphosphates (dNTPs) are incorporated into the polymerase chain reaction (PCR) products for the detection, and slab gel electrophoresis (with long track length polyacrylamide gels) is used in HDA.1,4,11,12 However, in the interest of higher efficiency detection, greater convenience, and safety, a few studies have evaluated the use of fluorescent dye-labeled primers or fluorescent dNTPs (instead of radioactive chemicals) for HDA via slab gel electrophoresis and capillary electrophoresis (CE).4,13,14 (2) Henson, D. E.; Srivastava S.; Kramer, B. S. Curr. Opin. Oncol. 1999, 11, 419-425. (3) Minamoto, T.; Mai, M.; Ronai, Z. Cancer Detect. Prev. 2000, 24, 1-12. (4) Nataraj, A. J.; Olivos-glander, I.; Kusukawa, N.; Highsmith, W. E., Jr. Electrophoresis 1999, 20, 1177-1185. (5) Arnold, N.; Gross, E.; Schwarz-Boeger, U.; Pfisterer, J.; Jonat, W.; Kiechle, M. Hum. Mutat. 1999, 14, 333-339. (6) Gross, E.; Arnold, N.; Goette, J.; Schwarz-Boeger, U.; Kiechle, M. Hum. Genet. 1999, 105, 72-78. (7) Struewing, J. P.; Hartge, P.; Wacholder, S.; Baker, S. M.; Berlin, M.; McAdams, M.; Timmerman, M. M.; Brody, L. C.; Tucker, M. A. N. Engl. J. Med. 1997, 336, 1401-1408. (8) Hacia, J. G.; Brody, L. C.; Chee, M. S.; Fodor, S. P.; Collins, F. S. Nat. Genet. 1996, 14, 441-447. (9) Gayther, S. A.; Harrington, P.; Russell, P.; Kharkevich, G.; Garkavtseva, R. F.; Ponder, B. A. Am. J. Hum. Genet. 1996, 58, 451-456. (10) Mansukhani, M. M.; Nastiuk, K. L.; Hibshoosh, H.; Kularatne, P.; Russo, D.; Krolewski, J. J. Diagn. Mol. Pathol. 1997, 6, 229-237. (11) White, M. B.; Carvalho, M.; Derse, D.; O’Brien, S. J.; Dean, M. Genomics 1992, 12, 301-306. (12) Markoff, A.; Sormbroen, H.; Bogdanova, N.; Preisler-Adams, S.; Ganev, V.; Dworniczak, B.; Horst, J. Eur. J. Hum. Genet. 1998, 6, 145-150. (13) Cheng, J.; Kasuga, T.; Mitchelson, K. R.; Lightly, E. R.; Watson, N. D.; Martin, W. J.; Atkinson, D. J. Chromatogr., A 1994, 677, 169-177. (14) Jackson, H. A.; Bowen, D. J.; Worwood, M. Br. J. Haematol. 1997, 98, 856859.

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Capillary electrophoresis (CE) offers several unique advantages over the traditional slab gel electrophoresis, the most important of which are the high speed, high resolution, small reagent consumption, and miniscule sample requirements.15 In recent years, microchip electrophoresis has emerged as an attractive platform for high-speed, high-throughput DNA analysis because parallel analysis can be achieved with multiple channels on a single microchip.16-26 DNA analysis by capillary and microchip electrophoresis has begun to emerge as an attractive alternative to traditional slab gel electrophoresis. The key to this has been the definition of a limited number of polymer networks (or entangled polymers) that are effective as a molecular sieving matrix for size-based separation. Polymers such as cellulose derivatives, linear poly(acrylamide) (LPA), poly(dimethylacryamide) (pDMA), poly(ethylene oxide) (PEO), poly(vinylpyrrolidone) (PVP), and poly(N-isopropylacrylamide)-g-poly(ethylene oxide) (PNIPAM-g-PEO) have been used as the sieving matrixes to separate the DNA molecules.27-39 Although Jackson et al.14 and Bowen et al.40 showed CE-based HDA could be used to detect point mutations in the HFE (HLAH) gene responsible for hemochromatosis via duplex generation, the nature of the polymer used as the sieving matrix was not disclosed. Equally important in DNA separations is the chemical nature of the capillary or microchannel surface, where the electroosmotic flow may need to be minimized or eliminated for effective (15) Oda, R. P.; Clark, R.; Katzmann, J. A.; Landers, J. P. Electrophoresis 1997, 18, 1715-1723. (16) Kopp, M. U.; Crabtree, H. J.; Manz, A. Curr. Opin. Chem. Biol. 1997, 1, 410-419. (17) Ocvirk, G.; Munroe, M.; Tang, T.; Oleschuk, R.; Westra, K.; Harrison, D. J. Electrophoresis 2000, 21, 107-115. (18) Li, J.; Kelly, J. F.; Chernushevich, I.; Harrison, D. J.; Thibault, P. Anal. Chem. 2000, 72, 599-609. (19) Ramsey, J. M.; Jacobson, S. C.; Knapp, M. R. Nat. Med. 1995, 1, 10931096. (20) Ramsey, J. M. Nat. Biotechnol. 1999, 17, 1061-1062. (21) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (22) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186. (23) Liu, S.; Shi, Y.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566-573. (24) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (25) Hofga¨rtner, W. T.; Hu ¨ hmer, A. F. R.; Landers, J. P.; Kant, J. A. Clin. Chem. 1999, 45, 2120-2128. (26) Huang, Z.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 5309-5314. (27) Carrilho, E. Electrophoresis 2000, 21, 55-65. (28) Nishimura, A.; Tsuhako, M.; Miki, T.; Ogihara, T.; Baba, Y. Chem. Pharm. Bull. (Tokyo) 1998, 46, 294-297. (29) Barron, A. E.; Sunada, W. M.; Blanch, H. W. Electrophoresis 1995, 16, 6474. (30) Shihabi, Z. K. J. Chromatogr., A 1999, 853, 349-354. (31) Ren, J.; Ulvik, A.; Refsum, H.; Ueland, P. M. Anal. Biochem. 1999, 276, 188-194. (32) Madabhushi, R. S. Electrophoresis 1998, 19, 224-230. (33) Madabhushi, R. S.; Vainer, M.; Dolnik, V.; Enad, S.; Barker, D. L.; Harris, D. W.; Mansfield, E. S. Electrophoresis 1997, 18, 104-111. (34) Kim, Y.; Yeung, E. S. Electrophoresis 1997, 18, 2901-2908. (35) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388. (36) Gao, Q.; Pang, H. M.; Yeung, E. S. Electrophoresis 1999, 20, 1518-1526. (37) Liang, D.; Song, L.; Zhou, S.; Zaitsev, V. S.; Chu, B. Electrophoresis 1999, 20, 2856-2863. (38) Song, L.; Fang, D.; Kobos, R. K.; Pace, S. J.; Chu, B. Electrophoresis 1999, 20, 2847-2855. (39) Liang, D.; Chu, B. Electrophoresis 1998, 19, 2447-2453. (40) Bowen, D. J.; Standen, G. R.; Granville, S.; Bowley, S.; Wood, N. A.; Bidwell, J. Thromb. Haemostasis 1997, 77, 119-122.

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separation. Although DNA separations have been successfully carried out in both coated and uncoated capillaries, the analysis of unpurified PCR products (containing salts and protein) has generally been carried out in the capillaries or microchannels with chemically modified (coated) surfaces.21,22,24,29,30,41 We previously reported that CE-based mutation detection via SSCP and HDA could be accomplished using hydroxyethylcellulose (HEC) as the polymer network and a commercial capillary having a fluorocarbon (FC)-modified surface.41a,b For microchipbased electrophoretic DNA analysis, polyacrylamide (PA)-derivatized surfaces based on a modified Hjerteń procedure42 have been the most commonly used, but require that the PCR sample be desalted in order to achieve good resolution and acceptable longevity.24,27 This may be the result of the chemical instability of the silica oxide bond (Si-O-Si, formed after the silanizing reagent reacted with the silanol group on the surface) under basic conditions.43 Moreover, there may be certain DNA separation conditions or applications where desalting of the PCR sample is detrimental. This is the case with CE-based heteroduplex analysis, where desalting the PCR products actually decreased the resolution.41b It is clear that a limited number of polymer choices and surfacecoating options are currently available for effective DNA separation under SSCP or HDA conditions by capillary electrophoresis. Additionally, if DNA mutation detection protocols are to be developed in microfabricated chips, novel coating/polymer combinations need to be defined. In this report, we compare and contrast, using capillary electrophoresis as the platform, the effectiveness of several coating and polymer combinations for detecting mutations in breast cancer susceptibility gene (BRCA1) via HDA. Using fluorescently labeled primers for amplification, laser-induced fluorescence for detection, and HEC as the sieving polymer, capillaries coated with either chlorodimethyloctylsilane (OCT)-PVP or (γ-methacryloxypropyl)trimethoxysilane (MET)PA were compared with commercial FC-coated capillaries for their effectiveness in detecting BRCA1 mutations by CE. EXPERIMENTAL SECTION Reagents. GeneAmp thin-walled PCR tubes, 10× PCR buffer, 25 mM MgCl2, 100 mM dNTPs stock solutions, and Taq DNA polymerase (5 units/µL) were from Perkin-Elmer (Norwalk, CT). Boric acid, ethylenediaminetetraacetic acid tetrasodium salt (EDTA), tris(hydroxymethyl)aminomethane (tris), ammonium persulfate (APS), and pBR322 HaeIII digest (982 µg/mL) were from Sigma Chemical Co. (St. Louis, MO). HEC [Mr 250 000, the viscosity of 2% HEC in H2O is 90-105 cP at 25 °C (supplier information)], MET, OCT, and chlorotrimethylsilane were from Aldrich Chemical Co. (Milwaukee, WI). Allyldimethylchlorosilane (ALLY), 4-chlorobutyldimethylchlorosilane (BUTYL), and 7-octenyltrimethoxysilane (OCTE) were from Gelest, Inc. (Tullytown, PA). Ethidium bromide, BODIPY FL propanol (4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propanol, 504 nm/510 nm), (41) (a) Tian, H.; Jaquins-Gerstl, A.; Munro, N. J.; Trucco, M.; Brody, L. C.; Landers, J. P. Genomics 2000, 63, 25-34. (b) Tian, H.; Brody, L. C.; Munro, N. J.; Landers, J. P. Genome Res. 2000, 10, 1403-1413. (42) Hjerteń, S. J. Chromatogr. 1985, 347, 191-198. (43) Nakatani, M.; Shibukawa, A.; Nakagawa, T. Electrophoresis 1995, 16, 14511456.

Table 1. Primers Used for Heteroduplex Analysis position

primers

Ta

Tm

size (bp)

185delAG (Exon 2, BRCA1)

forward: 5′-GAAGTTGTCATTTTATAAACCTTT-3′ reverse: 5′-TGTCTTTTCTTCCCTAGTATGT-3′

53 53

56

258

1294del40 (Exon 11, BRCA1)

forward: 5′-CCATGCTCAGAGAATCCTAG-3′ reverse: 5′-GATCACTGGCCAGTAAGTCT-3′

55 54

56

232

R1443G (4446C>G) (Exon 13, BRCA1)

forward: 5′-AGCTGTGTTAGAACAGCATG-3′ reverse: 5′-TGTTGGAGCTAGGTCCTTAC-3′

54 54

56

204

5382insC (Exon 20, BRCA1)

forward: 5′-ATATGACGTGTCTGCTCCAC-3′ reverse: 5′-CCTGTGTGAAAGTATCTAGCAC-3′

56 54

58

296

5677insA (Exon 24, BRCA1)

forward2: 5′-ATGAATTGACACTAATCTCTGC-3′ reverse1: 5′-CCACTTTGTAAGCTCATTCTT-3′

54 54

56

223

Figure 1. Heteroduplex analysis using the FC capillary and the HEC polymer as the sieving matrix. Panels I and II show the heteroduplex analysis results of the wild type and the heterozygous mutants, respectively. PCR conditions were described within the text. CE conditions were as follows: the FC-coated capillary was 50 µm (i.d.) by 27 cm (effective length 20 cm); PCR products were directly injected into the capillary for 20 s at 370 V/cm. The separation was carried out at 370 V/cm and the current was 27.0-27.6 µA, using the reversed polarity (inlet as cathode and outlet as anode). The capillary was maintained at 30 °C. LIF detection (em/ex: 520 nm/488 nm) was used. The separation buffer was 2.5% HEC (Mr 250 000) in 1×TBE buffer (pH 8.6) containing 10% glycerol. The peak with an asterisk (*) is wild-type homoduplex DNA fragment. The bracketed region is the duplex region of a mutant, containing homoduplex and heteroduplex DNA fragments.

and YO-PRO-1 were from Molecular Probes (Eugene, OR). Ultrapure formamide, N,N,N′,N′-tetramethylenediamine (TEMED), and 2-mL disposable polystyrene cuvettes were from Fisher Scientific, Inc. (Pittsburgh, PA). PVP (Mr 1 000 000, the viscosity of 1.5% PVP in 1×TBE buffer is 3.9 cP at 25 °C) and LPA (Mr 700 000-1 000 000) were from Polysciences, Inc. (Warrington, PA). Long Ranger gel solution was from FMC BioProducts (Rockland, ME). µSil-Fluorocarbon polymer (FC)-coated capillaries were from J&W Scientific, Inc. (Folsom, CA). Bare fused-silica

capillary (50 µm i.d., 365 µm o.d.) was from Polymicro Technologies (Phoenix, AZ). Capillary Coating Procedures. The coating procedures were modified from Hjerteń42 for coating PA and from Srinivasan et al.43 and Hofga¨rtner et al.25 for coating PVP. The details were as follows: Pretreatment. The bare fused-silica capillaries (50 µm i.d., 365 µm o.d.) were first washed with d.i. water for 10 min, rinsed with 1 M NaOH for 1 h, followed with d.i. water until the pH paper Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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indicated it was neutral, then dried at 105 °C for 1 h or rinsed with ethanol for 10 min, and dried at 80 °C for 1 h. In the case of coating the capillary with polyacrylamide, the bare fused-silica capillary was rinsed with the following solutions: d.i. water for 10 min, 1 M NaOH for 1 h, d.i. water for 10 min, 0.1 M HCl for 0.5 h, d.i. water until the pH paper indicated it was neutral, and then with anhydrous methanol for 20 min. Silanization. The pretreated capillary was filled with the specific silanizing reagent (OCT or others) in toluene (160 µL in 4 mL of anhydrous toluene), sealed with Teflon septa and stored at room temperature for 18 h, then rinsed with toluene, refilled with chlorotrimethylsilane in toluene (160 µL in 4 mL of anhydrous toluene), sealed with Teflon septa and stored at room temperature for another 18 h, and then rinsed with toluene, followed by rinsing with water. Polymerization. For coating PVP, 10 mL of 4% PVP polymer solution, prepared by dissolving 2 g of PVP (Mr 1 000 000) in 50 mL of 50 mM phosphate (pH 8.2), 10 µL of TEMED, and 100 µL of 10% freshly made ammonium persulfate (w/v in water) were mixed, degassed, and filled into the silanized capillary. The PVPfilled capillary (with Teflon septa) was then heated for 18 h in an oven at 80 °C. For coating PA, 10 mL of 3% Long Ranger gel solution (prepared by mixing 0.6 mL of 50% Long Ranger gel solution with 9.4 mL of 1×TBE (pH 8.2)), 50 µL of TEMED, and 50 µL of 10% freshly made ammonium persulfate (w/w in water) were mixed, degassed, and filled into the silanized capillary; the acrylamide-filled capillary was sealed by Teflon septa and stored at room temperature for 18 h. All the coated capillaries were rinsed with water for 1 h before use. Measuring the Electroosmotic Flow (EOF). The new capillary (50 µm i.d. by 27 cm, effective length 20 cm) was rinsed with the running buffer 1×TBE (pH 8.2) for 10 min before measuring EOF. BODIPY FL propanol (50 µM) was used as the neutral marker and hydrodynamically injected for 2 s at 0.5 psi. The separation was carried out at 556 V/cm using the normal polarity (outlet as cathode and inlet as anode). The capillary was maintained at 30 °C. Genomic DNA Isolation. Genomic DNA was isolated from lymphoblastoid cell lines obtained from the individuals heterozygous for the mutations in BRCA1 (Coriell Cell Repositories, Camden, NJ). All were used in an anonymous fashion in the study described. The concentrations of previously purified human genomic DNA were measured by PicoGreen dsDNA quantitation assay45 before use. The presence of BRCA1 mutations was confirmed by fluorescent dideoxy sequencing. Polymerase Chain Reaction. Primers used to flank the five mutations were from Friedman et al.46 or designed on the basis of the BRCA1 mRNA sequence on the Genome Database (http:// www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query, 1999), and the genomic sequences on the web site of the Breast Cancer Information Core (http://www.nhgri.nih.gov/Intramural_research/ Lab_transfer/Bic/, 1999); The primers were evaluated by the program on the following web site: http://www.williamstone.com/ primers/calculator/ and the estimate annealing temperatures for (44) Srinivasan, K.; Pohl, C.; Avdalovic, N. Anal. Chem. 1997, 69, 2798-2805. (45) Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P. Anal. Biochem. 1997, 249, 228-238. (46) Friedman, L. S.; Ostermeyer, E. A.; Szabo, C. I.; Dowd, P.; Lynch, E. D.; Rowell, S. E.; King, M. C. Nat. Genet. 1994, 8, 399-404.

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Figure 2. Effect of different coatings on capillary electrophoresisbased heteroduplex analysis. The PCR products were amplified from the heterozygous individual containing 1294del40 and analyzed under the same conditions shown in Figure 1 except that all the separation was carried out at 370 V/cm and the capillaries coated by the procedures described in the text were used. The EOF for each capillary was measured according to the procedures also described in the text. EOF of the bare fused-silica capillary was 3.7 × 10-4 cm2/ V‚s under the conditions used. The different silanizing reagents and polymers used for modifying the surface of the capillary were as follows: (A) CH2dC(CH3)COO(CH2)3Si(OCH3)3 (MET), PVP, EOF is 8.5 × 10-6 cm2/V‚s; 24.8 µA. (B) CH2dC(CH2)Si(CH3)2(Cl) (ALLYL), PVP, EOF is 8.9 × 10-6 cm2/V‚s; 25.0 µA. (C) Cl(CH2)4Si(CH3)2(Cl) (BUTYL), PVP, EOF is 19.7 × 10-6 cm2/V‚s; 25.5 µA. (D) CH2dCH(CH2)6Si(OCH3)3 (OCTE), PVP, EOF is 7.3 × 10-6 cm2/V‚s; 25.4 µA. (E) CH2dC(CH3)COO(CH2)3Si(OCH3)3 (MET), PA, EOF is 4.9 × 10-6 cm2/V‚s; 25.3 µA. (F) FC capillary (J&W), EOF is 6.7 × 10-6 cm2/ V‚s; 24.9 µA. (G) CH3(CH2)7Si(CH3)2(Cl) (OCT), PVP, EOF is 8.1 × 10-6 cm2/V‚s; 24.7 µA.

each primer are listed in Table 1. In all cases, one 6-FAM-tagged forward primer and one unlabeled reverse primer were used to amplify the DNA fragments (ordered from Life Technologies, Gaithersburg, MD). The sizes of the DNA fragments amplified for detecting each mutation are included in Figure 1. PCR amplifications of BRCA1 alleles were carried out in a Progene thermocycler (Techne, Princeton, NJ) with the following reagents in 50-µL reaction mixtures: 40-80 ng of genomic DNA, 0.2 µM concetrations of the appropriate primers (one is 6-FAM tagged for HDA), 1 mM dNTPs, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, and 5.0 units of AmpliTaq polymerase. Each PCR reaction mixture was heated for 5 min at 95 °C, followed by 35 cycles of 1 min at 94 °C, 0.5 min at the annealing temperature as in Tables 1, and 0.5 min at 72 °C. A final 10-min extension at 72 °C was used following the final temperature cycle.

Figure 3. Comparison of MET-PA and OCT-PVP capillaries for heteroduplex analysis. Panels I and II show the heteroduplex analysis results using MET-PA and OCT-PVP, respectively. The PCR products were analyzed under the conditions described in Figure 1 except that the capillary was MET-PA or OCT-PVP. Currents were as follows: left panel, 25.3-25.7 µA; right panel, 24.5-24.7 µA. The bracketed region is the duplex region of a mutant, containing homoduplex and heteroduplex DNA fragments. The bracketed region with an asterisk (*) contains two homoduplex DNA fragments. The bracketed region with a # contains two heteroduplex DNA fragments.

CE-Based Heteroduplex Analysis. For mutation detection by HDA, a Beckman P/ACE 5510 system (Fullerton, CA) with the P/ACE LIF detector [with the excitation at 488 nm (an argon ion laser) and the emission at 520 nm] was used. Capillary electrophoresis conditions were as follows: the capillary was 50 µm (i.d.) by 27 cm (effective length 20 cm); the separation buffer was 2.5% (w/v) HEC in 1×TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA, pH 8.6 unless specified) containing 10% glycerol. The PCR products were introduced into the capillary by electrokinetical injection for 20 s at 370 V/cm. The separation was carried out at 370 V/cm using the reversed polarity (inlet as cathode and outlet as anode), and the capillary was maintained at 30 or 20 °C. RESULTS AND DISCUSSION HDA is one of the most commonly used methods for screening unknown mutations.4 The principles underlying mutation detection using HDA are based on conformational differences with duplex DNA (dsDNA). Wild-type dsDNA consists of two complementary strands (homoduplex) while the dsDNA from the heterozygous individual contains two complementary strands (wild-type homoduplex and mutant homoduplex) and two mismatched strands (two heteroduplexes, so-called bulge or bubble dsDNA47). The goal of HDA is simplesdiscriminate the homoduplex DNA from the heteroduplex DNA fragments on the basis of their conforma-

tions under native conditions. Since HDA is generally accomplished via slab gel electrophoresis,4 there is great interest in defining electrophoretic methods that are less labor-intensive. For the purposes of microchip electrophoresis method development, mutations in the breast cancer susceptibility genes, BRCA1 have been utilized as a model system. More than 600 mutations have been identified in BRCA1 and BRCA2 genes, with an unusually large proportion of these being deletion and insertion mutations47b these are typically easier to detect by conventional electrophoretic detection methods than substitution (point) mutations, especially via heteroduplex analysis.4 However, it is important that a detection method be capable of detecting all three types of DNA mutations. Heteroduplex Analysis Using a Commercial Coated Capillary and HEC as Polymer. As a result of our previous work on mutation detection with SSCP, we were able to define a fast, simple CE-based HDA method for mutation detection, using HEC as the sieving matrix in a commercially available FC-coated capillary, where only three steps [DNA purification, DNA amplification by PCR, and PCR products analysis (HDA)] are needed for detection of heterozygous mutations.41b As shown in Figure 1, wild type and (47) (a) Bhattacharyya, A.; Lilley, D. M. J. Nucleic Acid Res. 1989, 17, 68216840. (b) Hussain, S. P.; Harris, C. C. Cancer Res 1998, 58, 4023-37.


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Figure 4. Effect of different coatings on the analysis of dsDNA fragments. The capillary was 50 µm (i.d.) by 27 cm (the effective length was 20 cm) with the following coating: (A) OCT-PVP, 9.5 µA; (B) FC-coated capillary, 9.3 µA; (C) MET-PVP, 7.6 µA. The DNA sample, pBR 322 HaeIII digest (0.982 µg/mL), was injected electrokinetically for 4 s at 370 V/cm. The separation field was 148 V/cm with the capillary maintained at 20 °C. The separation buffer was 1.5% HEC in 1×TBE containing 0.5 µM YO-PRO-1. Other CE conditions were as in Figure 1. The number of the theoretical plates for each capillary was calculated according to the equation, N ) 5.54(L/W1/2)2, using the peak of 267 bp in the electropherograms.

five heterozygous mutants can be identified in less than 10 min by CE based on the heteroduplex profiles. The wild type is represented by a single peak (marked with an asterisk in the left box, panel I) while the heterozygous mutants are represented by several peaks in the duplex region (bracketed regions in panel II). The results in Figure 1 are similar to those obtained by traditional slab gel electrophoresis, where two or three bands could be observed in the duplex regions with a polyacrylamide gel or a conformation-sensitive gel.1,4,11,12 While the FC coating of capillaries is clearly effective for HDA, the proprietary nature of this coating complicates transfer to microchip structures. Comparison of Different Capillary Coatings for HDA. Numerous chemical methods have been utilized for minimizing the effect of surface silanol groups on the capillary wall, therefore, suppressing the EOF and decreasing the interactions between the analytes and the capillary wall. This is especially so for the CE separation of proteins15 and DNA.27 Gao et al.35,36 demonstrated that PVP not only sufficed as a polymer for DNA separation but also functioned to dynamically coat the capillary surfaces. While the usefulness of PVP for CE-based DNA separations was confirmed in our laboratory, it was found to be ineffective for microchip analysis, possibly due to the roughness of the channel surface on the microchip.48 As mentioned in the introduction, the PA coating pioneered by Hjerteń42 has been widely used for DNA 5488 Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

analysis. Hjerteń42 employed a two-step coating procedure by using a bifunctional silanizing reagent to react with the silanol groups on the surface, followed by in situ polymerization of the monomer containing a vinyl group. He concluded the presence of a polymerizable CdC group was essential in both the monomer and the silanizing reagent for coupling. However, our experience with PA-coated microchannels is that they have limited longevity with PCR product analysis if the samples are not desalted. Srinivasan et al.44 took a different approach and used a “hydrogen abstraction mechanism” to attach the polymer to the surface of the silanized capillary, where the silanizing reagent did not contain a vinyl group and cross-linking of the polymers with the surface was achieved by the free-radical condition.49 On the basis of the above mechanisms, five different silanizing reagents were tested: MET, ALLY, BUTYL, OCTE, and OCT. Three of these (MET, ALLYL, OCTE) are bifunctional silanizing reagents (containing CdC group) and were chosen to mask the silanol groups on the surface of the capillary wall. As far as the polymeric coating is concerned, one polymer (PVP) and one monomer (acrylamide) were chosen to be cross-linked with the silanized surface. (48) Munro, N. J.; Snow, K.; Kant, J. A.; Landers, J. P. Clin. Chem. 1999, 45, 1906-1917. (49) Anderson, C. C.; Rodriguez, F.; Thurston, D. A. J. Appl. Polym. Sci. 1979, 23, 2453-2462.

Figure 5. Comparison of different capillaries with different EOFs for heteroduplex analysis. (A) and (B) show the heteroduplex analysis results with the 185delAG and 1294del40 mutants, using FC, OCT-PVP, and OCT-PA capillaries with EOF as 6.7 × 10-6, 8.1 × 10-6, and 3.77 × 10-6 cm2/V‚s, respectively. The separation buffer consisted of 2.5% HEC and 10% glycerol in 1×TBE. Other CE conditions were as in Figure 1. Currents were as follows: (a) 23.0, (b) 24.6, (c) 24.1, (d) 22.3, (e) 24.7, and (f) 22.7 µA.

The effectiveness of the different coatings was determined in two ways: (1) by measuring the EOF (see Experimental Section) and (2) by using the coated capillaries for heteroduplex analysis. Figure 2 shows the results of analyzing one heterozygous BRCA1 mutant using capillaries coated with different silanizing reagents and polymers and how they compare with the commercial FCcoated capillary. In general, the EOF was found to be 19-76-fold lower than that observed with bare fused-silica capillary. Interestingly, the MET-PA coating had the lowest EOF (4.9 × 10-6 cm2/ V‚s) but the OCT-PVP coating provided the best resolution for detecting the heterozygous mutant, 1294del40. As seen in Figure 2G, two homoduplexes, two heteroduplexes, and two singlestranded DNA fragments were resolved. Comparison of the MET-PA and OCT-PVP Capillary Coatings. Since MET-PA is a widely utilized coating for DNA analysis, but the OCT-PVP provided the best resolution, at least for HDA detection of the 1294del40 mutation, we furthered the comparison of these for detecting other BRCA1 mutations. This was accomplished using BRCA1 mutations, 185delAG, 1294del40, 4446C>G, 5382insC, and 5677insA, which are categorized as deletion, substitution, and insertion mutations. As illustrated in Figure 3, using the OCT-PVP coating provided the resolution in the duplex region that was, at the very least, as good as that obtained using MET-PA (panels A-C) and in some cases (panels D, E) provided better resolution. To obtain a more quantitative information of the MET-PA and OCT-PVP coatings, efficiency calculations were carried out with a dsDNA fragment standard (pBR 322 HaeIII digest) separated

in the MET-PA-, OCT-PVP-, an FC-coated capillaries (using 1.5% HEC). As can be seen in Figure 4, the 123- and 124-bp fragments were partially resolved in the OCT-PVP-coated and FC-coated capillaries (Figure 4B). The number of the theoretical plates (N) was calculated for each coating according to the standard equation [N ) 5.54(L/W1/2)2], using the migration time (L) and half width (W1/2) of the 267-bp peak in the electropherograms in Figure 4. Under the same separation conditions, the number of the theoretical plates was 9.67 × 105, 7.53 × 105, and 2.09 × 105 for OCTPVP, FC, and MET-PA coating, respectively. With these three coatings under these separation conditions, the efficiency of the OCT-PVP coating was roughly 5-fold better than with the METPA coatingsthis was in agreement with the results of HDA. It is noteworthy that we also tested the effectiveness of the OCT-PVP for coating a glass microchip. Preliminary experiments showed OCT-PVP coating was also effective for coating the microchannels for microchip-based heteroduplex analysis (data not shown). Details associated with a more thorough evaluation of the OCT-PVP-coated microchip will be reported elsewhere.50 Comparison of the OCT-PA and OCT-PVP Capillary Coatings. On the basis of the data in Figures 2-4, one could argue that the use of the appropriate silanizing reagent (OCT or MET in this case) was of prime importance and that the polymer seemed less critical to successfully coating the surface for HDA (comparing OCT-PVP, MET-PVP, and MET-PA). In addition, one would assume that the magnitude of EOF reduction, reflecting (50) Munro et al. Robust polymeric microchannel coatings for microchip-based analysis of neat, PCR products in preparation.


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Figure 6. Identification of homoduplexes, heteroduplexes, and single-stranded DNA fragments in the electropherograms for CE-based mutation detection. In all cases, the FC-coated capillary, 50 µM (i.d.) by 27 cm (effective capillary length, 20 cm) was used, the injection time was 30 s at 370 V/cm, the separation was carried out at 148 V/cm, and the separation temperature was 20 °C. (A) PCR products were injected directly, the separation buffer was 1.5% HEC in 1×TBE (pH 8.6), 8.2 µA; (B) 1 µL of the PCR products mixed with 10 µL of deionized formamide and 0.5 µL of 0.3 M NaOH was denatured at 95 °C for 5 min, cooled on ice for 5 min before injection; 8.4 µA; (C) PCR products were injected directly, the separation buffer was 1.5% HEC in 1×TBE containing 1 µM ethidium bromide, 8.7 µA; (D) PCR products were injected directly, the separation buffer was 1.5% HEC containing 0.5 µM YO-PRO-1, 8.0 µA. Other conditions were the same as in Figure 1. The bracketed region with # contains two heteroduplex DNA fragments.

the degree to which the surface silanol groups have been masked, would also result in a more effective HDA. The latter is not born out in the data from Figure 2. While the MET-PA coating was associated with an EOF (4.9 × 10-6 cm2/V‚s) that was almost half that of the OCT-PVP (8.1 × 10-6 cm2/V‚s), the resolution for HDA and for resolving a dsDNA fragment standard was inferior to OCT-PVP. It is noteworthy that we also tested the coupling of OCT with acrylamide monomer as a surface coating (OCT-PA). With a reduction in EOF to 3.77 × 10-6 cm2/V‚s, this coating provided even lower EOF than MET-PA. The heteroduplex analysis results obtained with the OCT-PA coating for two deletion mutations (1294del40, 185delAG) are shown in Figure 5. Comparing the results obtained using this coating with those obtained with OCT-PVP- and FC-coated capillaries, it is obvious that the OCT-PA coating provided the poorest resolution. The possible reasons for this may include stability considerations with the polyacrylamide coating and/or nonhomogeneity of the coated surface. The former is evidenced by the observation that EOF tripled after 10 runs. Collectively, these results indicate that, while reduced EOF may be required for high-resolution dsDNA separations, the magnitude of EOF reduction is not the only parameter for gauging the effectiveness of the coated surface for HDA. Not surprising, the physical and chemical character of the polymer 5490


covalently attached to an adequate bifunctional reagent is of paramount importance. Peak Identification in Heteroduplex Analysis Profiles. In CE-based heteroduplex analysis, dsDNA (both homoduplexes and heteroduplexes) can be identified by including the dsDNA intercalator in the separation buffer, where the detectability of the dsDNA will be greatly enhanced, with ssDNA barely enhanced, if at all.51,52 Two methods can be used to identify the ssDNA fragments in the heteroduplex profiles shown in the previous figures. One approach is to use SSCP where, under denaturing conditions, the PCR products can be distinguished by their intensities, ssDNA fragments are generally increased.41a Alternatively, an intercalator (e.g., ethidium bromide) added to the separation buffer allows for the intensities of both dsDNA and ssDNA to be enhanced.53 Figure 6 shows that peak identification in HDA profiles can be accomplished. As a result of the 40-bp difference between the wild type and mutant DNA fragments, the two homoduplexes (192 and 232 bp) are easily identified by comparing the migration times (51) Skeidsvoll, J.; Ueland, P. M. Anal. Biochem. 1995, 231, 359-365. (52) Suzuki, T.; Fujikura, K.; Higashiyama, T.; Takata, K. J. Histochem. Cytochem. 1997, 45, 49-53. (53) Rye, H. S.; Glazer, A. N. Nucleic Acids Res. 1995, 23, 1215-1222.

Figure 7. Comparison of different polymers for capillary electrophoresis-based heteroduplex analysis. Panels I and II show the heteroduplex analysis results with the wild type and the heterozygous mutant, 1294del40. The PCR products were injected electrokinetically for 30 s at 370 V/cm. Detection was mediated by laser-induced fluorescence (em/ex: 520 nm/488 nm). The polymer-coated capillary and the separation voltage used were as follows: (A) 2.5% LPA containing 10% glycerol in 1×TBE (pH 8.6), FC capillary (27 cm × 50 µm), 370 V/cm, 17.0-17.2 µA; (B) 2.5% HEC containing 10% glycerol in 1×TBE (pH 8.6), FC capillary (37 cm × 50 µm), 486 V/cm, 34.7-35 µA; (C) 2.5% HEC containing 10% glycerol in 1×TBE (pH 8.6), OCT-PVP capillary (37 cm × 50 µm), 486 V/cm, 31.9-32.0 µA; (D) 6.0% PVP containing 10% glycerol in 1×TBE (pH 8.6), OCT-PVP capillary (37 cm × 50 µm), 486 V/cm, 27.6-28.4 µA; (E) 6.0% PVP in 1×TBE (pH 8.6), OCT-PVP capillary (37 cm × 50 µm), 486 V/cm, 25.3-25.7 µA.

with those in pBR 322 HaeIII digest (Figure 6A,D). On the basis of the results of SSCP analysis (Figure 6B), and separations that included ethidium bromide (Figure 6C) and YO-PRO-1 (Figure 6D) in the separation buffer, the two small peaks (at 13.2 and 13.8 min) in the Figure 6A (mt) heteroduplex profile can be assigned as the 192- and 232-bp ssDNA fragments. Similarly, the two partially resolved peaks bracketed in Figure 6A (mt) (around 14.1 min) can be assigned as the two heteroduplexes. Comparing Figure 6C with Figure 6A, it is clear that including ethidium bromide in the separation buffer improves the resolution of the two heteroduplexes in the 1294del40 mutant, where all six DNA fragments are baseline resolved. This approach for improving resolution with heteroduplex analysis was also reported previously by Cheng et al.13 Comparison of Different Polymers for Heteroduplex Analysis. Having defined the OCT-PVP coating as optimal for providing resolution for mutation detection via HDA, a number of different polymers were evaluated for comparison with HEC, including LPA and PVP. The six DNA peaks that were observed in the heterozygous deletion mutant (1294del40) in Figure 2G were not resolved with 2.5% LPA/10% glycerol (Figure 7A, right

panel) or with 5% LPA/10% glycerol (data not shown). Although the wild type and the mutant could be discriminated based on the heteroduplex profiles using 6% PVP, with or without 10% glycerol, the identity of these peaks is complicated, especially when more than six peaks were observed using PVP and glycerol (shown in Figure 7D). As shown in Figure 7C, the best resolution for detecting 1294del40 mutant was obtained by using 2.5% HEC and the OCT-PVP coating, which is also the best combination for detecting the other four mutants as shown in Figure 3. Experiments demonstrated that a higher concentration of HEC or lower separation voltage could certainly improve the resolution for heteroduplex analysis. Reproducibility of Heteroduplex Analysis and EOF. Using the OCT-PVP-coated capillary and a 1×TBE solution containing 2.5% HEC and 10% glycerol, the migration times of the three peaks in the duplex region of 185delAG were determined. Nineteen injections using the same capillary yielded migration times (and coefficient of variance values) for the three DNA fragments of 7.56 ( 0.253 (CV ) 3.35%, n ) 19), 7.80 ( 0.264 (CV ) 3.38%, n ) 19), and 8.34 ( 0.288 min (CV ) 3.46%, n ) 19). This is acceptable reproducibility for the separation of a PCR-amplified Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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DNA sample in a coated capillary. As far as the EOF is concerned, reproducibility was less impressive with the coated capillaries than would be expected from the migration time data above. Using a free solution protocol to measure the EOF, values for the bare fused-silica, FC-coated and OCT-PVP-coated capillaries (in cm2/ V‚s) were 3.71 × 10-4 ( 0.07 × 10-4 (CV ) 1.8%, n ) 3, same capillary), 7.8 × 10-6 ( 1.1 × 10-6 (CV ) 14.7%, n ) 4, same capillary), and 9.8 × 10-6 ( 2.2 × 10-6 (CV ) 22.5%, n ) 4, different capillaries), respectively. The fact that the reproducibility of HD analysis was better than that observed with the EOF measurements indicates, again, that reduction of EOF is not a dominating factor in effective heteroduplex analysis. A more detailed discussion on capillary and microchip coating for reduction of EOF can be found elsewhere.50

impressive. However, it is not clear why the combination of the OCT-PVP coating with HEC polymer network provides the optimal conditions for CE-based mutation detection via HDA. It may be due to the appropriate pore size provided by the HEC in combination with the uniform character and/or stability of the OCT-PVP coating. It is interesting, but not overly surprising, that lower EOF is not the sole prerequisite for higher efficiency for DNA separations. Assuming that the BRCA1 mutations represent a reasonable model system for HDA, these data show that optimal detection of mutations is obtained with an OCT-PVP-coated capillary and 2.5% HEC as a polymeric sieving matrix. As with other applications developed in this laboratory, these conditions should be exportable to the microchip format for high-speed and high-throughput heteroduplex analysis.

CONCLUSIONS By exploring a number of silanizing reagents, coating polymers, and polymer networks, it was possible to identify the ideal combination for effective CE detection of mutations in the breast cancer susceptibility gene, BRCA1. Of the five silanizing reagents, three coating polymers, and three polymer networks evaluated, the OCT-PVP coating in combination with HEC as a polymer network appears to be optimal for the HDA application. Not only was the performance optimum, based on resolution of the mutant DNA fragments in comparison to the MET-PA so commonly used, but also its resilience and longevity for repeated use was

ACKNOWLEDGMENT We thank Biomedical Research Support Facilities, School of Medicine, University of Pittsburgh, for providing DNA sequencing services, and Beckman Instruments for the equipment. We also thank Dr. Andreas F. R. Hu¨hmer for his efforts in the initial coating work and Nicole J. Munro for helpful discussions.

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Received for review May 1, 2000. Accepted August 28, 2000. AC0004916

Effective Capillary Electrophoresis-Based Heteroduplex Analysis

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