Authors
Anne-Leila Meistertzheim, Isabelle Calves, Sébastien Artigaud, Carolyn S. Friedman, Christine Paillard, Jean Laroche, and Claude Ferec
Summary
This protocol permits the mutation scanning of PCR products by high-resolution DNA melting analysis requiring the inclusion of a saturating intercalating dye in the PCR mix without labelled probe. During a scanning process, fluorescent melting curves of PCR amplicons are analyzed. Mutations modifying melting curve shapes, are allowed to be further characterized by sequencing because melting is not destructive. The method detects on small amplicons (120pb), 100% of heterozygous and 75% of homozygous variants in a single step. Homozygous variants are detected in a second time at 100% by adding wild-type reference DNA in the tube. For mitochondrial haplotypes, homozygous variants are discriminated by increased the size of the amplicons (700pb). HRMA can be conducted without knowledge on number of mutations, small insertions or deletions of the studied sequence in non-model species. The method accomplishes simultaneous gene scanning in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires <1h20 (96- or 384-well plates) and melting acquisition takes 10 min per plate.Further details
The protocol was published on Protocol Exchange on 15 May 2012. To see the entire protocol, click on the source link.Advertisement
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