A novel RFLP-ARMS TaqMan PCR-based method for detecting the BRAF V600E mutation in melanoma

Oncology Letters
Yunqing ZhangJie Huang

Abstract

To enable the rapid and sensitive screening of the BRAF V600E mutation in clinical samples, a novel method combining restriction fragment length polymorphism (RFLP) analysis with the popular amplification refractory mutation system (ARMS) TaqMan quantitative (qPCR) genotyping method in a single reaction tube was developed. A total of 2 primer pairs were designed to enrich for and genotype the BRAF mutational hotspot (RFLP primers and ARMS primers) and a restriction enzyme was used to remove the wild-type alleles. The analysis revealed that this method detected mutant alleles in mixed samples containing >0.1% mutant sequences. In a survey of 53 melanoma samples, this method detected 21 mutation-positive samples. This novel RFLP-ARMS TaqMan qPCR protocol may prove useful for detecting mutations in clinical samples containing only a small proportion of mutant alleles.

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Methods Mentioned

BETA
PCR
biopsies
genotyping

Software Mentioned

DNASTAR
ABI7300
SPSS

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