High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrrolidone) solution.
Single-nucleotide polymorphism detection has been the focus of much attention recently. Although many methods have been reported, low-cost, high-throughput, and high-detection-rate methods are still in demand. We present a fast and reliable mutation detection scheme based on temperature-gradient capillary electrophoresis. A large temperature gradient (10 degrees C) was applied with a precision of 0.02 degrees C and a temperature ramp of 0.7 degrees C/min. Multiple unlabeled samples from PCR were injected and analyzed. Ethidium bromide was used as the intercalating dye for laser-induced fluorescence detection. Mutations can be recognized by comparing the electrophoretic patterns of the heteroduplex with that of a homoduplex reference without prior knowledge of the exact type of mutation present. Mutations in all five test samples were successfully detected with high confidence. This scheme is demonstrated in 96-capillary array electrophoresis for screening single-point polymorphism in large numbers of samples prior to full sequencing of only the positive samples to identify the nature of the mutation.
Gao
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USDOE and Department of Chemistry, Iowa State University, Ames 50011, USA.
Yeung
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ES
D004247:DNA / Q000032:analysis / Q000235:genetics; D019075:Electrophoresis, Capillary; D017354:Point Mutation; D011110:Polymorphism, Genetic; D011205:Povidone
D004247:DNA; D011205:Povidone