A improved tetra-primer amplification refractory mutation system PCR based assay to detect four single nucleotide polymorphisms associated with folate metabolism in a single reaction

Abstract

Objective To develop a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) assay for detecting four single nucleotide polymorphisms (SNPs): rs1801133, rs1801131, rs1805087 and rs1801394 associated with folate metabolism in a single reaction tube. Methods Methodology was developed. Then, it applied the established method to analyze 150 physical examination children′s anticoagulant blood samples admitted to the Department of Clinical Laboratory, Children′s Hospital of Hebei Province from January 2017 to April 2017. Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs1801133, rs1801131, rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy. A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions. The amplified products were analyzed by QIAxcel capillary electrophoresis, and the corresponding SNP genotypes of 150 samples were identified. Furthermore, all samples were verified by direct sequencing. And the Hardy-Weinberg Equilibrium ( HWE) testing of four SNPs from 150 samples were conducted by SPSS17.0 Chi-square test. Results The improved T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours. Four SNPs of 150 whole blood samples were accurately classified and the results were completely consistent with direct sequencing. All the genotype frequencies of these four SNPs were in HWE (χrs18011332=0.69, Prs1801133=0.40; χrs18011312=0.21, Prs1801131=0.64; χrs18050872=3.32, Prs1805087=0.07; χrs18013942=1.91, Prs1801394=0.17). Conclusions The proposed improved T-ARMS-PCR combined with capillary electrophoresis in this study can accurately verify four SNPs associated with folate metabolism in a single reaction tube. This method might be a valuable tool to specifically guide the folate supplement in general population.(Chin J Lab Med, 2017, 40: 799-804) Key words: Folic acid; Metabolism; Polymorphism, single nucleotide; Polymerase Chain reaction; Electrophoresie, capillary; DNA mutational analysis; DNA primers