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Denaturing high‐performance liquid chromatography (DHPLC) compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presence of a mutation by the differential retention of homo‐ and heteroduplex DNA on reversed‐phase chromatography supports under partial denaturation. Temperature determines sensitivity, and its optimum can be predicted by computation. Single‐nucleotide substitutions, deletions, and insertions have been detected successfully by on‐line UV or fluorescence monitoring within 2–3 minutes in unpurified amplicons as large as 1.5 Kb. Sensitivity and specificity of DHPLC consistently exceed 96%. These features and its low cost make DHPLC one of the most powerful tools for the re‐sequencing of the human and other genomes. Aside from its application to the mutational analysis of candidate genes, DHPLC has proven instrumental in …