PCR-RFLP法检测MTRR基因A66G多态位点的改进  

Improvement of PCR-RFLP method for detecting MTTR gene A66G polymorphism

作　　者：何震宇 顾取良 陈瑜丽 HE Zhenyu;GU Quliang;CHEN Yuli(School of Life Sciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangzhou,Guangdong,China,510006)

机构地区：[1]广东药科大学生命科学与生物制药学院,广东广州510006

出　　处：《分子诊断与治疗杂志》2021年第10期1701-1704,共4页Journal of Molecular Diagnostics and Therapy

基　　金：广东省科技计划项目(公益研究与能力建设专项资金项目)(2015A030401098)。

摘　　要：目的对现有检测MTRR基因A66G多态位点的PCR-RFLP法进行改进,避免因Nde I酶切不完全可能导致的基因型误判。方法设计1对PCR引物,上游引物3′端倒数第3位含错配碱基,下游引物结合位置位于一个天然的Nde I识别序列(CATATG)72 nt之后,使用该对引物扩增包含MTRR基因A66G多态位点的靶序列,将多态位点相关的原序列AATRTG(R为多态位点碱基A/G)变为CATRTG,以便通过限制性核酸内切酶Nde I消化来甄别多态位点碱基具体为A还是G,多态位点下游315 nt处含有天然的Nde I识别序列作为内对照酶切位点,PCR产物用Nde I消化制作限制性酶切图谱以判断基因型。结果PCR可成功扩增预期大小为478 bp的靶片段,在用Nde I消化PCR产物制作而成的限制性酶切图谱中,凭借小于PCR产物的2种特征性酶切条带可明确判断各样品基因型,即便酶切不完全即出现PCR产物残留或酶切中间产物时,基因型的判断也不受干扰,分型结果得到基因测序支持。使用该法检测了100例样品,检得MTRR基因A66G多态位点3种基因型即AA、AG、GG的频率分别为0.57、0.34、0.09;A和G等位基因频率分别为0.74和0.26,符合Hardy-Weinberg遗传平衡(X^(2)=1.355,P=0.508)。结论本方法由于内对照酶切位点的引入,能克服传统PCR-RFLP法使用Nde I酶检测MTRR基因A66G多态位点酶切不完全可能导致的基因型误判。Objective To improve the existing PCR-RFLP method for detecting A66G polymorphic locus of MTRR gene to avoid genotype misjudgment that may be caused by incomplete Nde I digestion.Methods A pair of primers for PCR was designed.The 3′-terminal antepenultimate of the forward primer contains mismatched base,and the binding position of the downstream primer is 72 nt behind a natural Nde I recognition sequence(CATATG).The target sequence of A66G polymorphic locus of MTRR gene was amplified by this pair primers.The polymorphic locus-related original sequence AATRTG(R=A/G)was changed into CATRTG,in order to use the restriction endonuclease Nde I digestion to identify whether the polymorphic base is A or G,an intrinsic Nde I recognition sequence CATATG at 315 nucleotides downstream of the polymorphic locus was used as the internal control.PCR products were digested by NdeⅠto produce restriction enzyme digestion maps to identify genotypes.Results The target fragment of 478 bp was amplified by PCR,the restriction enzyme digestion map was made from the PCR products digested with Nde I.The genotype of each sample can be identified by the characteristic bands smaller than the PCR products in the restriction enzyme digestion map.Even if the digestion is incomplete,the residue of the PCR products or the intermediate products of the digestion can help to determine the genotype.the genotyping method was furtherconfirmed by gene sequencing.100 samples were detected.The frequencies of the three genotypes of MTRR gene A66G polymorphism,namely AA,AG,and GG were 0.57,0.34,and 0.09,respectively.The frequencies of A and G alleles were 0.74 and 0.26,respectively,in line with the Hardy-Weinberg genetic balance(X^(2)=1.355,P=0.508).Conclusion Due to the introduction of endogenous control restriction site,this method can overcome the genotype misjudgment that may be caused by the incomplete restriction of the A66G polymorphic site of the MTRR gene using the Nde I enzyme in the traditional PCR-RFLP method.

关 键 词：甲硫氨酸合成酶还原酶 基因分型 聚合酶链反应-限制性片段长度多态性 

分 类 号：R440[医药卫生—诊断学]