应用SNP敏感性分子开关检测线粒体DNA编码区单核苷酸多态性的研究

发布时间：2018-03-08 07:47

本文选题：SNP　切入点：等位基因特异性PCR　出处：《南京医科大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的建立高保真DNA聚合酶介导的硫化修饰等位基因特异性PCR(allele-specific PCR, AS-PCR),并将其应用于线粒体DNA(mitochondrial DNA, mtDNA)编码区单核苷酸多态性(single nucleotide polymorphism, SNP)的研究工作,探讨其实际应用价值;同时调查60例中国江苏无关汉族个体mtDNA编码区4个SNP基因座等位基因频率和单倍型分布情况,为群体遗传学提供新的数据。方法 1根据线粒体DNA编码区10400(T/C)位点设计未修饰及3′末端硫化修饰的等位基因特异性引物,采用低保真Taq和高保真Pfu DNA聚合酶进行引物延伸反应,2%琼脂糖凝胶电泳比较扩增结果。 2从线粒体DNA编码区筛选多态性较好的4个SNP(C12705T、G8701A、G8584A、C10400T)基因座。针对每个SNP基因座设计两条片段相差4个碱基的3′末端硫化修饰等位基因特异性引物和一条公共引物。血液标本采自中国江苏地区60名汉族无关、健康个体。采用Chelex-100法提取模板mtDNA,利用Pfu高保真DNA聚合酶进行扩增反应。扩增产物经聚丙烯酰胺凝胶电泳、银染显带后分析样本的基因型并应用直接测序技术验证分型结果的准确性。结果 1与传统AS-PCR相比,高保真Pfu DNA聚合酶介导的硫化修饰等位基因特异性PCR独具的开关效应使引物延伸的特异性大大提高,降低了SNP分析的假阳性率,提高了对SNP检测的准确性和可靠性。 2应用上述方法对江苏地区汉族群体60名无关个体4个mtDNA-SNP基因座进行了准确分型,不同等位基因型各为一条长度不同的单一谱带,分型结果与直接测序完全一致。4个mtDNA-SNP基因座C12705T、G8701A、G8584A、C10400T等位基因频率分别为0.37/0.63、0.47/0.53、0.83/0.17、0.53/0.47,共检出6种单倍型,单倍型的基因多样性为0.7438,偶合概率值为0.2686。结论本研究成功建立了由高保真DNA聚合酶和3′末端硫化修饰等位基因特异性引物共同构成的SNP敏感性分子开关技术,并应用于mtDNA编码区单核苷酸多态性的群体遗传学研究,证实该技术检测SNP的准确性和优越性,具有极高的临床实用价值,值得在基因SNP研究中进一步推广;同时为线粒体SNP基因座在法医学等方面的应用提供了方法学和群体遗传学基础。
[Abstract]:Purpose. A high fidelity DNA polymerase mediated sulphide modified allele-specific PCR(allele-specific PCR, AS-PCRN, was established and applied to the study of single nucleotide polymorphisms (SNPs) in the coding region of mitochondrial DNA(mitochondrial DNA (mtDNA), and its practical application value was discussed. The allelic frequency and haplotype distribution of 4 SNP loci in the mtDNA coding region of 60 unrelated Han individuals from Jiangsu Province of China were also investigated in order to provide new data for population genetics. Method. 1 the allele specific primers of unmodified and 3 '-terminal vulcanization modification were designed according to the 10400 T / C site of mitochondrial DNA coding region. The amplification results were compared by using low fidelity Taq and high fidelity Pfu DNA polymerase for primer extension reaction of 2% agarose gel electrophoresis. 2 four SNPs C12705TnG8701AG8584AG10400T loci were screened from the mitochondrial DNA coding region. A 3'terminal sulphide modified allele specific primer and a common blood primer were designed for each SNP locus. The liquid samples were collected from 60 Han nationality in Jiangsu, China. The template mtDNA was extracted by Chelex-100 method and amplified by Pfu high fidelity DNA polymerase. The amplified products were analyzed by polyacrylamide gel electrophoresis. The genotypes of the samples were analyzed after silver staining and the accuracy of the typing results was verified by direct sequencing. Results. 1 compared with traditional AS-PCR, the unique switch effect of high fidelity Pfu DNA polymerase mediated sulphide modified allele-specific PCR greatly improved the specificity of primer extension and reduced the false positive rate of SNP analysis. The accuracy and reliability of SNP detection are improved. (2) the four mtDNA-SNP loci of 60 unrelated individuals in the Han population of Jiangsu province were accurately typed by the above method. The genotypes of each allele were a single band with different lengths. The allele frequencies of four mtDNA-SNP loci C12705TnG8701AnG8584AH10400T were 0.37 / 0.630.47 / 0.530.83 / 0.170.53 / 0.47, respectively. Six haplotypes were detected, the genetic diversity of haplotypes was 0.7438, and the probability of coupling was 0.2686. Conclusion. In this study, a novel SNP sensitive molecular switch technique, composed of high fidelity DNA polymerase and 3 'terminal vulcanized modified allele specific primers, was successfully established and applied to the population genetics of single nucleotide polymorphisms in the mtDNA coding region. The accuracy and superiority of this technique in detecting SNP are proved to be of high clinical and practical value. It is worthy to be further popularized in the study of gene SNP. It also provides methodology and population genetic basis for the application of mitochondrial SNP locus in forensic science.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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