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Correct heteroduplex formation for mutation detection analysis
M J Smith1, K E Humphrey, R Cappai
1Department of Pathology, The University of Melbourne, Melbourne.
Background:
The majority of mutation detection methods for unknown mutations are polymerase chain reaction (PCR)-based methods dependent on the formation 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 technique and other methods reliant on heteroduplex formation. It was found that the size and sequence context of the fragment being investigated inhibited correct heteroduplex formation. The problem was overcome by dividing the sequence into two overlapping fragments.
Conclusions:
Early identification of this problem in other fragments will help with the rapid optimization of PCR-based mutation detection methods.