Denaturing High-Performance Liquid Chromatography

Denaturing high performance liquid chromatography (dHPLC) is a fast and reliable technique for the DNA variation screening (1 ,2 ).It can detect in minutes with close to 100% sensitivity and specificity single-base substitutions as well as small deletions and insertions in DNA fragments ranging from 80-1500 base pairs in size (3 ,4 ).In partially denaturing HPLC, typically 2–10 chromosomes are compared as a mixture of PCR products. Upon mixing, denaturing and reannealing of amplicons containing one or more mismatches, not only the original homoduplices are formed again but, simultaneously, the sense and anti-sense strands of either homoduplex form two heteroduplices. Heteroduplices denature more extensively at elevated column temperatures in the range of 48-67�C; they are retained less on the chromatographic separation matrix, allowing the separation of homo- and heteroduplex species by ion-pair reversed-phase HPLC (IP-RP-HPLC) (5 ). Characteristic peak patterns both for homozygous and heterozygous samples are obtained.