Open AccessDetecting Rare Thrombophilia Variants by High-Resolution Melting
Author(s) -
Michael Seipp,
Carl T. Wittwer
Publication year - 2011
Publication title -
clinical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.705
H-Index - 218
eISSN - 1530-8561
pISSN - 0009-9147
DOI - 10.1373/clinchem.2010.159160
Subject(s) - high resolution melt , genotyping , amplicon , melting curve analysis , methylenetetrahydrofolate reductase , genetics , microbiology and biotechnology , factor v leiden , factor v , thrombophilia , biology , genotype , polymerase chain reaction , medicine , gene , venous thrombosis , pregnancy , thrombosis
To the Editor:Genotyping by amplicon melting is a minimalistic approach to genotyping, requiring only PCR, a fluorescent DNA dye, and single-color acquisition of a melting curve in a closed tube. Multiplex genotyping for the F5 1 [coagulation factor V (proaccelerin, labile factor)] Leiden (1691G>A), F2 [coagulation factor II (thrombin)] 20210G>A, and MTHFR [methylenetetrahydrofolate reductase (NAD(P)H)] 1298A>C and 677C>T thrombophilia mutations distinguishes up to 81 (34) combinations in solution without physical processing (1). Furthermore, we previously suggested that it might be possible to detect additional rare variants within the sequences of the quadruplex amplicons by a combination of melting temperature ( T m) and shape discrimination enabled by high-resolution melting analysis. Amplified heterozygotes have a complex melting curve shape consisting of 2 homoduplexes (wild type and mutant) and 2 heteroduplexes that are influenced by the class of single-nucleotide polymorphism and other variants …
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