Improved efficiency of mutation detection by denaturing high-performance liquid chromatography using modified primers and hybridization procedure

The addition of a 20-base CC-clamp to a DNA fragment enabled mutations to be detected by denaturing highperformance liquid chromatography (DHPLC) in the higher melting domain of the two-domain fragment DYS271, The mutations were undetectable in the absence of the CC-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 degreesC, whereas mutations in the low melting domain were detected at 56 degreesC, Computer modeling of the melting behavior agreed well with the experimental results, facilitating computer design of DHPLC amplicons,