Yang Siqi, Shi Huiting, Zhang Zhanfa, Chen Zhuochen, Yang Weiming, Zhu Weifeng
The Second Clinic Medical College, and Nanchang University, Nanchang, China.
College of Basic Medical Sciences, Nanchang University, Nanchang, China.
Genet Test Mol Biomarkers. 2022 Jan;26(1):43-48. doi: 10.1089/gtmb.2021.0145.
The standard methods for tri-allelic single nucleotide polymorphism (SNP) genotyping require special equipment and are costly to perform. The aim of this study was to establish a fast, simple, and low-cost method to differentiate among tri-allelic SNPs in general laboratories. Based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) typing of bi-allelic SNPs, we developed a penta-primer amplification refractory mutation system-polymerase chain reaction (P-ARMS-PCR) method characterize tri-allelic SNPs. The two most studied tri-allelic SNPs, rs2032582 and rs3091244, were genotyped using P-ARMS-PCR in 110 volunteers, and the results were verified by direct DNA sequencing. For rs2032582, there were 20 samples (18.18%) with the GG genotype, 3 (2.73%) with the AA genotype, 24 (21.82%) with the TT genotype, 43 (39.09%) with the GT genotype, 11 (10.00%) with the AG genotype, and 9 (8.18%) with the AT genotype. For rs3091244, there were 67 samples (60.91%) with the CC genotype, 1 (0.91%) with the AA genotype, 8 (7.27%) with the CT genotype, 31 (28.18%) with the CA genotype, and 3 (2.73%) with the AT genotype. The genotypic distributions of rs2032582 ( = 0.482) and rs3091244 ( = 0.492) were in Hardy-Weinberg equilibrium. The DNA sequencing results were entirely consistent with the results of P-ARMS-PCR. P-ARMS-PCR is an accurate, rapid, simple, and low-cost characterization method for tri-allelic SNP genotyping of rs2032582 and rs3091244.
三等位基因单核苷酸多态性（SNP）基因分型的标准方法需要特殊设备，且执行成本高昂。本研究的目的是在普通实验室中建立一种快速、简单且低成本的方法来区分三等位基因SNP。基于双等位基因SNP的四引物扩增阻滞突变系统-聚合酶链反应（T-ARMS-PCR）分型，我们开发了一种五引物扩增阻滞突变系统-聚合酶链反应（P-ARMS-PCR）方法来鉴定三等位基因SNP。使用P-ARMS-PCR对110名志愿者中研究最多的两个三等位基因SNP，即rs2032582和rs3091244进行基因分型，并通过直接DNA测序验证结果。对于rs2032582，有20个样本（18.18%）为GG基因型，3个（2.73%）为AA基因型，24个（21.82%）为TT基因型，43个（39.09%）为GT基因型，11个（10.00%）为AG基因型，9个（8.18%）为AT基因型。对于rs3091244，有67个样本（60.91%）为CC基因型，1个（0.91%）为AA基因型，8个（7.27%）为CT基因型，31个（28.18%）为CA基因型，3个（2.73%）为AT基因型。rs2032582（=0.482）和rs3091244（=0.492）的基因型分布处于哈迪-温伯格平衡。DNA测序结果与P-ARMS-PCR结果完全一致。P-ARMS-PCR是一种用于rs2032582和rs3091244三等位基因SNP基因分型的准确、快速、简单且低成本的鉴定方法。
