title: Allele-specific, non-extendable primer blocker PCR (AS-NEPB-PCR) for DNA mutation detection in cancer.
abstract: Allele-specific amplification, combined with TaqMan probe real-time polymerase chain reaction (real-time AS-PCR), has been widely used for detecting genetic variants, single nucleotide polymorphisms, and genetic mutations. In addition, several probe-blocking methods have been introduced in real-time AS-PCR to block amplification of wild-type templates and to increase detection sensitivity and specificity. However, most of these methods provide a limited sensitivity of no better than 1% and are complex in design of blockers, and thus cannot be readily adapted for different mutation assays. We have developed a modified non-extendable primer blocker (NEPB) for real-time AS-PCR (AS-NEPB-PCR). The NEPB method provides an easy design of allele-specific primer and corresponding primer blocker that can be used in any single nucleotide polymorphism or mutation detection, specifically in the detection of low-frequency mutations. The method is straight-forward in assay optimization and can achieve 0.1% sensitivity with 100% specificity (95% confidence interval, 92-100%) in detecting K-Ras, B-Raf, and EGFR mutations in cancer cells.
authors.affiliation: Veridex/Ortho-Clinical Diagnostics, Johnson & Johnson, Raritan, New Jersey, USA.
authors.forename: Haiying
authors.initials: H
authors.lastname: Wang
authors.forename: John
authors.initials: J
authors.lastname: Jiang
authors.forename: Bianca
authors.initials: B
authors.lastname: Mostert
authors.forename: Anieta
authors.initials: A
authors.lastname: Sieuwerts
authors.forename: John W M
authors.initials: JW
authors.lastname: Martens
authors.forename: Stefan
authors.initials: S
authors.lastname: Sleijfer
authors.forename: John A
authors.initials: JA
authors.lastname: Foekens
authors.forename: Yixin
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D018773:Genes, erbB-1; D011905:Genes, ras; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D048493:Proto-Oncogene Proteins B-raf