title: Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.
abstract: 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.
authors.affiliation: Department of Pathology, UUMC, 5B418, 50 N. Medical Drive, Salt Lake City, Utah 84105, USA.
authors.forename: Jesse
authors.initials: J
authors.lastname: Montgomery
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: Robert
authors.initials: R
authors.lastname: Palais
authors.forename: Luming
authors.initials: L
authors.lastname: Zhou
mesh_terms: D016000:Cluster Analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D005453:Fluorescence; D005821:Genetic Techniques; D005838:Genotype; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D009841:Oligonucleotides