Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons

Clinical Chemistry
Michael LiewCarl Wittwer

Abstract

High-resolution melting of PCR amplicons with the DNA dye LCGreen I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (</=50 bp). Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and beta-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees C). Although mo...Continue Reading

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Citations

Apr 2, 2013·Indian Journal of Clinical Biochemistry : IJCB·Ankur Shah, Claire Seedhouse
Jun 15, 2007·Pharmacogenomics·Gudrun H ReedCarl T Wittwer
Feb 25, 2014·Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics·Gustavo Nuñez-AcuñaCristian Gallardo-Escárate
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