Development of a Multiplex Tetra-Primer Amplification Refractory Mutation System PCR to Detect Three Single Nucleotide Polymorphisms Associated with Breast Cancer

Abstract

We describe a multiplex tetra-primer amplification refractory mutation system PCR (M-T-ARMS PCR) for detecting three single nucleotide polymorphisms associated with breast cancer, including rs4784227(C>T), rs12196489(G>A) and rs3803662(T>C). Three sets of chimeric primers consisting of a specific sequence fused to a universal sequence at the 5′ end were used in one single PCR reaction, followed by a capillary electrophoresis to separate PCR products and genotyping. We applied this method to test 70 samples from whole blood and mouth swab specimens to demonstrate the performance. All mutations were accurately detected and results were confirmed by direct DNA sequencing. M-T-ARMS PCR coupled with capillary electrophoresis is a simple and accurate assay that allows the simultaneous detection of three mutations in a single tube.