Open AccessCombining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000
Author(s) -
Jonci N. Wolff,
Neil J. Gemmell
Publication year - 2008
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112719
Subject(s) - taqman , allele , nuclease , biology , single nucleotide polymorphism , microbiology and biotechnology , minor allele frequency , genetics , variants of pcr , snp , allele frequency , restriction enzyme , snp genotyping , polymerase chain reaction , dna , genotype , gene
TaqMan-nuclease assays are widely used for the qualitative detection of single nucleotide polymorphisms (SNPs) and the determination of biallelic states in pooled or heterozygous DNA samples. These assays are highly specific, reproducible, and suitable for high-throughput approaches. A crucial limitation of this method, and others, is the detection of minor allele frequencies with detection limits of generally 3% to 9% for minor allele contributions. Here we describe the combination of customized TaqMan-nuclease assay and allele-specific restriction to increase the sensitivity of this method, allowing the qualitative detection of allele contributions as low as 0.05%.