Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis

This protocol permits the simultaneous mutation scanning and genotyping of PCR products by high-resolution DNA melting analysis. This is achieved using asymmetric PCR performed in the presence of a saturating fluorescent DNA dye and unlabeled oligonucleotide probes. Fluorescent melting curves of both PCR amplicons and amplicon-probe duplexes are analyzed. The shape of the PCR amplicon melting transition reveals the presence of heterozygotes, whereas specific genotyping is enabled by melting of the unlabeled probe amplicon duplex. Unbiased hierarchal clustering of melting transitions automatically groups different sequence variants; this allows common variants to be easily recognized and genotyped. This technique may be used in both laboratory research and clinical settings to study single-nucleotide polymorphisms and small insertions and deletions, and to diagnose associated genetic disorders. High-resolution melting analysis accomplishes simultaneous gene scanning and mutation genotyping in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires < 30 min (capillaries) or 1.5 h (96- or 384- well plates) and melting acquisition takes 1-2 min per capillary or 5 min per plate.