Protocol abstract
Nature Protocols 2, - 59 - 66 (2007)
Published online: 22 February 2007 | doi:10.1038/nprot.2007.10
Subject Categories: Genetic analysis | Nucleic acid based molecular biology
Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis
Jesse Montgomery1, Carl T Wittwer1, Robert Palais1 & Luming Zhou1
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.
- Department of Pathology, UUMC, 5B418, 50 N. Medical Drive, Salt Lake City, Utah 84105, USA.
Correspondence to: Carl T Wittwer1 e-mail: carl.wittwer@path.utah.edu
nature-products
A-Z product listing
- 10
3'-blocked forward direction oligonucleotide probe(Idaho Technology Inc.) - 10 dNTP mix(Idaho Technology)
- 10 Forward direction oligonucleotide primer(Idaho Technology Inc.)
- 10 LCGreen Plus(Idaho Technology Inc.)
- 10 PCR buffer(Idaho Technology Inc.)
- 10 Reverse direction oligonucleotide primer(Idaho Technology Inc.)