Improved Efficiency of Mutation Detection by Denaturing High-Performance Liquid Chromatography Using Modified Primers and Hybridization Procedure

Abstract

The addition of a 20-base GC-clamp to a DNA fragment enabled mutations to be detected by denaturing high-performance liquid chromatography (DHPLC) in the higher melting domain of the two-domain fragment DYS271. The mutations were undetectable in the absence of the GC-clamp. The heteroduplex yield was greatly decreased by the presence of mutations in the high melting domain, presumably because this region anneals first during cooling, leading to selection of the more stable homoduplexes. Suppression of sequence-dependent melting behavior using betaine increased the heteroduplex yield almost four-fold. Mutations in the high melting domain were detected at 60°C, whereas mutations in the low melting domain were detected at 56°C. Computer modeling of the melting behavior agreed well with the experimental results, facilitating computer design of DHPLC amplicons.