应用复合MS-PCR法快速检测线粒体DNA编码区单核苷酸多态性的研究

发布时间：2019-01-29 05:47

【摘要】： 目的：为提高线粒体DNA(mitochondrial DNA，mtDNA)遗传标记的个人识别能力和非母排除能力，筛选线粒体DNA编码区频率较好的单核苷酸多态(single nucleotide polymorphism SNP)基因座，利用复合MS-PCR(multiplexed mutagenically separated PCR)技术、银染分型，建立线粒体DNA编码区10个SNP基因座复合扩增分型系统，以期为法医鉴定提供mtDNA-SNP新的遗传标记和新的检测手段，并探讨其应用价值；同时调查160例中国成都无关汉族个体mtDNA编码区10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：从线粒体DNA编码区筛选多态性较好的10个SNP(T12705C，G8701A，A8584G，T10400C，T4883C，C10873T，A15301G，C14783T，A3010G，A5460G)基因座。针对每个SNP基因座分别设计两条片段相差3～4个碱基的等位基因特异性引物和一条公共引物，优化扩增条件，使10个SNP基因座同时在一管中扩增，PCR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。应用直接测序技术以T12705C、C10873T和A5460G基因座为例研究该方法检测的准确性。结果：本研究成功建立了10个mtDNA-SNP基因座复合扩增，聚丙烯酰胺凝胶电泳、银染显带分型的方法；不同SNP基因座为长度不同的单一谱带，其分型结果与直接测序一致；用上述方法对成都汉族160名无关个体10个mtDNA-SNP基因座进行了准确分型，分型结果可靠，重复性好。10个mtDNA-SNP基因座C12705T、A8701G、G8584A、C10400T，，T4883C、C10873T、A15301G、C14783T、A3010G、A5460G等位基因频率分别为0．382／0．618、0．482／0．518、0．825／0．175、0．494／0．506、0．169／0．831、0．513／0．487、0．538／0．462、0．506／0．494、0．163／0．837、0．09／0．91，共检出18种单倍型，单倍型的基因多样性为0．8617，偶合概率值为0．1437。结论：本研究通过自行设计的10套引物，成功的构建了10个线粒体SNP基因座复合MS-PCR扩增、银染分型体系。为提高mtDNA-SNP基因座的个人识别能力和非母排除能力提供了一种简单、快速、准确、经济、有效的SNP分型新方法；对建立线粒体DNA-SNP数据库，为线粒体SNP基因座在考古学、进化学和法医学个人识别和亲子鉴定提供了方法学和群体遗传学基础。
[Abstract]:Objective: to improve the ability of individual identification and non-maternal exclusion of mitochondrial DNA (mitochondrial DNA,mtDNA (mtDNA) markers, and to screen single nucleotide polymorphic (single nucleotide polymorphism SNP) loci with better frequency of mitochondrial DNA coding region. A multiplex amplification typing system for 10 SNP loci of mitochondrial DNA coding region was established by using compound MS-PCR (multiplexed mutagenically separated PCR) technique and silver staining typing, in order to provide a new mtDNA-SNP genetic marker and a new detection method for forensic identification. And discusses its application value; The alleles frequency and haplotype distribution of 10 SNP loci in the mtDNA coding region of 160 unrelated Han individuals in Chengdu, China, were also investigated in order to provide new data for population genetics. Methods: ten polymorphic SNP loci (T12705CnG8701A, T10400CnT4883T10873T10873TC14783TC14783TFA3010GnA5460G) were screened from the mitochondrial DNA coding region. Two allele-specific primers and one common primer were designed for each SNP locus. The amplification conditions were optimized, and 10 SNP loci were amplified in one tube at the same time. The PCR products were analyzed by polyacrylamide gel electrophoresis and silver staining. The accuracy of this method was studied by using direct sequencing technique with T12705CnC10873T and A5460G loci as examples. Results: ten mtDNA-SNP loci were successfully amplified, polyacrylamide gel electrophoresis and silver staining banding. Different SNP loci were single bands of different length, and the typing results were consistent with direct sequencing. Using the above method, 10 mtDNA-SNP loci of 160 unrelated individuals in Chengdu Han nationality were accurately typed. The results of typing were reliable and reproducible. Ten mtDNA-SNP loci C12705TnA8701GGN G8584AC10400 T10400 T10883 C10873TUA15301GC14783TA3010G, C14783TA3010G. The allele frequency of A5460G is 0.382 / 0.618 / 0.482 / 0.518 / 0.825 / 0.175 / 0.494 / 0.506n 0.169r0.831 / 0.5130.538 / 0.462n 0.506 / 0.494r0.1630.163 / 0.830.09 / 0.991, respectively. A total of 18 haplotypes were identified. The genetic diversity of haplotypes was 0.8617 and the probability of coupling was 0.1437. Conclusion: in this study, 10 mtDNA loci were successfully amplified by 10 sets of primers designed by ourselves, and the silver staining typing system was constructed. It provides a simple, rapid, accurate, economical and effective method for SNP typing in order to improve the ability of individual recognition and non-maternal exclusion of mtDNA-SNP loci. The establishment of mitochondrial DNA-SNP database provides methodology and population genetic basis for mitochondrial SNP locus identification in archaeology, advanced chemistry and forensic medicine.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

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