Maria Erali, Karl V. Voelkerding, Carl T. Wittwer
Experimental and Molecular Pathology
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 saturating DNA dyes that distinguish many different species. For example, mutation scanning (identifying heterozygotes) by melting is closed-tube and has similar or superior sensitivity and specificity compared to methods that require physical separation. With high resolution melting, SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Highly polymorphic HLA loci can be melted to establish sequence identity for transplantation matching. Simultaneous genotyping with one or more unlabeled probes and mutation scanning of the entire amplicon can be performed at the same time in the same tube, vastly decreasing or eliminating the need for re-sequencing in genetic analysis. High resolution PCR product melting is homogeneous, closed-tube, rapid (1â5Â min), non-destructive and does not require covalently-labeled fluorescent probes. In the clinical laboratory, it is an ideal format for in-house testing, with minimal cost and time requirements for new assay development.
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