Optimisation of a DHPLC Method for Mutation and Polymorphism Detection

This application note by Cecil Instruments describes a method for Denaturing HPLC (DHPLC), an ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). DHPLC identifies mutations and polymorphisms based on detection of heteroduplex formation between mismatched nucleotides in double-stranded PCR amplified DNA. Sequence variation creates a mixed population of heteroduplexes and homoduplexes during reannealing of wildtype and mutant DNA. When this mixed population is analysed by HPLC under partially denaturing temperatures, the heteroduplexes elute from the column earlier than the homoduplexes because of their reduced melting temperature. Optimisation of DHPLC, requires the generation of a melting profile for each PCR fragment to determine its optimal temperature for analysis.