Correct heteroduplex formation for mutation detection analysis

Background: The majority of mutation detection methods for unknown mutation s are polymerase chain reaction (PCR)-based methods dependent on the format ion of heteroduplexes between wild-type and mutant strands of DNA. Methods and Results: This report discusses the difficulties associated with forming heteroduplexes with a large DNA fragment and the implications for subsequent mutation detection by the chemical cleavage of mismatch techniqu e and other methods reliant on heteroduplex formation. It was found that th e size and sequence context of the fragment being investigated inhibited co rrect heteroduplex formation. The problem was overcome by dividing the sequ ence into two overlapping fragments. Conclusions: Early identification of this problem in other fragments will h elp with the rapid optimization of PCR-based mutation detection methods.