Carl T Wittwer
University of Utah, United States of America
Separation of the two strands of DNA with heat (melting) is a fundamental property of DNA that is conveniently monitored with fluorescence. Conventional melting is performed after PCR on any real-time instrument to monitor product purity (dsDNA dyes) and sequence (hybridization probes). Recent advances include high-resolution instruments and heteroduplex detecting DNA dyes that distinguish many different species. Consensus is building that high resolution melting is the better mutation scanning technique than methods that require physical separation, such as dHPLC. Most, but not all, single base variants and small insertions or deletions can also be genotyped by amplicon melting. More complex regions can be typed with unlabeled probes or snapback primers. Mutation scanning and genotyping with one or more unlabeled probes can be performed at the same time in the same tube, eliminating most resequencing needs. The melting curves of highly polymorphic regions (such as HLA) can be melted to establish sequence identity without specific genotyping. High-resolution DNA melting is homogeneous, closed-tube, rapid (1-10 min), non-destructive and does not require fluorescent probes or real-time PCR.
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