A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

Chen Zhang,Hongwei Shen,Ying Liu,Kai Nie,Xuejun Ma,Mengjie Yang,Brian Z Ring,Xiyang Wu,Miao Wang,Gayle E Woloschak

doi:10.1371/journal.pone.0062126

Abstract

BackgroundThe tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP's, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs.MethodsA total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.ResultsOf the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.ConclusionsThis novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.

Highlights

The role of single nucleotide polymorphisms (SNP’s) in contributing to the variability between individuals in susceptibility to cancer[1], tumor growth and metastasis rate[2,3,4], as well as in treatment efficacy and adverse drug responses, has been well recognized[5,6]
We describe a novel multiplex T-ARMS-PCR allowing for the simultaneous genotyping of 6 SNPs associated with breast and gynecologic cancers in a single tube using 24 chimeric primers and a pair of universal primers The use of chimeric primers and a temperature switch PCR (TSP) strategy were combined with T-ARMS-PCR to optimize the amplification parameters and improve the throughput of SNP genotyping
A total of 186 samples were typed with a multiplex T-ARMSPCR assay and typed in parallel with direct sequencing to assess the accuracy and efficiency of the assay

Summary

Introduction

The role of single nucleotide polymorphisms (SNP’s) in contributing to the variability between individuals in susceptibility to cancer[1], tumor growth and metastasis rate[2,3,4], as well as in treatment efficacy and adverse drug responses, has been well recognized[5,6]. Among the many methods that have been developed to genotype SNPs, the tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) has proved to be rapid, simple and economical [7,8,9,10,11]. The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP’s, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification.

Methods

Results

Conclusion