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.
Montgomery
Jesse
J
Department of Pathology, UUMC, 5B418, 50 N. Medical Drive, Salt Lake City, Utah 84105, USA.
Wittwer
Carl T
CT
Palais
Robert
R
Zhou
Luming
L
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
D009841:Oligonucleotides