Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks.
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, (gamma-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylprrolidone) (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 FC-coated 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.
Tian
H
H
Department of Chemistry, University of Pittsburgh, Pennsylvania 15260, USA.
Brody
L C
LC
Mao
D
D
Landers
J P
JP
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D020180:Heteroduplex Analysis / Q000379:methods*; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D011108:Polymers; D013499:Surface Properties
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011108:Polymers; D004247:DNA