PloS one · 2013
A novel multiplex tetra-primer ARMS-PCR for the simultaneous genotyping of six single nucleotide polymorphisms associated with female cancers.
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Zhang Chen, Liu Ying, Ring Brian Z, Nie Kai, Yang Mengjie, Wang Miao, Shen Hongwei, Wu Xiyang, Ma Xuejun
Аннотация
BACKGROUND: 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. We assessed this process in the simultaneous genotyping of four breast cancer-and two cervical cancer risk-related SNPs.
METHODS: A 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.
RESULTS: Of 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.
CONCLUSIONS: This 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.
METHODS: A 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.
RESULTS: Of 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.
CONCLUSIONS: This 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.