武汉汉族群体24个mtDNA编码区SNPs研究
发布时间：2019-06-08 14:33
【摘要】：研究背景法医案件的现场生物检材,常因复杂环境因素的影响,核DNA容易发生降解,导致核DNA遗传标记检测阴性。此外,犯罪现场遗留的常见检材,例如:毛发,牙齿,骨头等,只含有少量DNA,分型检测难度较大。所以,当核基因组DNA的信息无法获得时,检测线粒体DNA(mtDNA)成为一常用的替代选择。单个细胞中mtDNA拷贝数远比核DNA多,而且线粒体基因组碱基突变率约是核DNA的10倍。尤其位于非编码区,HV-I区和HV-II区含有密集的多态性位点。控制区中所存在的大量碱基序列差异使其成为进行个人识别的主要方法。mtDNA具有母系遗传和缺乏重组的特性,具有相对较高的随机匹配概率。大样本调查中发现大约84%的个体拥有唯一的HV-I和HV-II mtDNA序列,但仍有部分个体的序列相同。利用mtDNA多态性作个人识别的意义在于排除同一性。群体遗传学研究发现,mtDNA特征是可以按血缘关系分簇,或分成单倍群。单倍群是以分布于编码区的特异性SNP位点来区分的,某些单倍群在特定人群中可呈分布广泛。虽然高变区含有较多的单倍群,但在15,447 bp的编码区也有相当数量的多态性位点。由于编码区承受的选择压力相对较大,因此这区段碱基序列的突变率更低,分析编码区的多态性位点成为法医线粒体DNA研究领域中,提高排除率的一种有用工具。 目的基于成熟的SNaPshot技术(微测序技术)建立一套简便、经济、高效的实验方案,调查武汉地区汉族人群mtDNA编码区24个SNPs的频率分布,为法医学领域的相关应用提供依据。 方法选择多态性较好的24个mtDNA-SNPs,查明其碱基序列,使用相关软件设计目的片段扩增和单碱基延伸的两套引物,首先利用等位基因特异性PCR技术扩增不同基因座的等位基因片段,然后使用纯化的PCR产物进行SBE反应,最后通过荧光检测识别所有基因座延伸的单个碱基类型,以此构建24个mtDNA-SNP基因座的荧光复合扩增检测体系,并用建立的检测体系对100名武汉汉族无关个体进行检测分型。 结果构建了24个mtDNA-SNP基因座荧光复合扩增体系,分型检测结果表现为:各组SNP基因座具有颜色异同的八个产物峰,不同SNP基因座的片段大小不同,同一SNP基因座的不同碱基表现出峰型位置的差异。在检测的100个样本中,发现31个单倍型,其频率范围是0.01~0.14。24个mtDNA-SNP基因座在武汉汉族群体中的单倍型多样性为0.952。 结论本研究所创建的mt-SNP复合扩增体系具有简便、经济、高效的特点,具有较高的法医学应用价值。
[Abstract]:Background the field biological samples of forensic cases are often prone to degradation of nuclear DNA due to the influence of complex environmental factors, which leads to the negative detection of nuclear DNA genetic markers. In addition, the common materials left over from the crime scene, such as hair, teeth, bones, etc., contain only a small amount of DNA, typing. Therefore, when the information of nuclear genomic DNA is not available, the detection of mitochondrial DNA (mtDNA) has become a common alternative. The number of copies of mtDNA in a single cell is much more than that of nuclear DNA, and the base mutation rate of mtDNA is about 10 times higher than that of nuclear DNA. Especially in the non-coding region, HV-I region and HV-II region contain dense polymorphism sites. A large number of base sequence differences in the control region make it the main method for personal identification. MtDNA has the characteristics of maternal inheritance and lack of recombination, and has a relatively high random matching probability. In the large sample survey, about 84% of the individuals had unique HV-I and HV-II mtDNA sequences, but some of them still had the same sequences. The significance of using mtDNA polymorphism as personal identification is to exclude identity. Population genetics studies have found that mtDNA can be grouped according to consanguinity or divided into haploids. Haploids are distinguished by specific SNP loci distributed in the coding region, and some haploids can be widely distributed in specific populations. Although the hypervariable region contains more haploids, there are also a considerable number of polymorphism loci in the coding region of 15447 bp. Because the selection pressure of the coding region is relatively high, the mutation rate of the base sequence in this region is lower. The analysis of the polymorphism sites in the coding region has become a useful tool to improve the exclusion rate in the field of forensic mitochondrial DNA. Objective to establish a simple, economical and efficient experimental scheme based on mature SNaPshot technology (microsequencing) to investigate the frequency distribution of 24 SNPs in mtDNA coding region of Han population in Wuhan, so as to provide evidence for the application of forensic science. Methods 24 mtDNA-SNPs, with good polymorphism were selected to identify their base sequences, and two sets of primers were designed to amplify the target fragment and extend the single base by using the relevant software. First, the allelic fragments of different loci were amplified by allele-specific PCR, then the purified PCR products were used for SBE reaction, and finally the single base types extending from all loci were identified by fluorescence detection. The fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed, and the typing of 100 unrelated individuals in Wuhan Han nationality was detected by the established detection system. Results the fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed. the results of typing showed that there were eight product peaks with different colors in each group of SNP loci, and the fragments of different SNP loci were different. The peak position of different bases at the same SNP locus was different. Of the 100 samples tested, 31 haplotypes were found, the frequency range of which was 0.01 鈮，
本文编号：2495362
[Abstract]:Background the field biological samples of forensic cases are often prone to degradation of nuclear DNA due to the influence of complex environmental factors, which leads to the negative detection of nuclear DNA genetic markers. In addition, the common materials left over from the crime scene, such as hair, teeth, bones, etc., contain only a small amount of DNA, typing. Therefore, when the information of nuclear genomic DNA is not available, the detection of mitochondrial DNA (mtDNA) has become a common alternative. The number of copies of mtDNA in a single cell is much more than that of nuclear DNA, and the base mutation rate of mtDNA is about 10 times higher than that of nuclear DNA. Especially in the non-coding region, HV-I region and HV-II region contain dense polymorphism sites. A large number of base sequence differences in the control region make it the main method for personal identification. MtDNA has the characteristics of maternal inheritance and lack of recombination, and has a relatively high random matching probability. In the large sample survey, about 84% of the individuals had unique HV-I and HV-II mtDNA sequences, but some of them still had the same sequences. The significance of using mtDNA polymorphism as personal identification is to exclude identity. Population genetics studies have found that mtDNA can be grouped according to consanguinity or divided into haploids. Haploids are distinguished by specific SNP loci distributed in the coding region, and some haploids can be widely distributed in specific populations. Although the hypervariable region contains more haploids, there are also a considerable number of polymorphism loci in the coding region of 15447 bp. Because the selection pressure of the coding region is relatively high, the mutation rate of the base sequence in this region is lower. The analysis of the polymorphism sites in the coding region has become a useful tool to improve the exclusion rate in the field of forensic mitochondrial DNA. Objective to establish a simple, economical and efficient experimental scheme based on mature SNaPshot technology (microsequencing) to investigate the frequency distribution of 24 SNPs in mtDNA coding region of Han population in Wuhan, so as to provide evidence for the application of forensic science. Methods 24 mtDNA-SNPs, with good polymorphism were selected to identify their base sequences, and two sets of primers were designed to amplify the target fragment and extend the single base by using the relevant software. First, the allelic fragments of different loci were amplified by allele-specific PCR, then the purified PCR products were used for SBE reaction, and finally the single base types extending from all loci were identified by fluorescence detection. The fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed, and the typing of 100 unrelated individuals in Wuhan Han nationality was detected by the established detection system. Results the fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed. the results of typing showed that there were eight product peaks with different colors in each group of SNP loci, and the fragments of different SNP loci were different. The peak position of different bases at the same SNP locus was different. Of the 100 samples tested, 31 haplotypes were found, the frequency range of which was 0.01 鈮，
本文编号：2495362
本文链接：https://www.wllwen.com/falvlunwen/fanzuizhian/2495362.html
