线粒体DNA序列多态性及其在法医学应用的研究

发布时间：2018-05-11 11:41

本文选题：线粒体 + DNA(mtDNA)　；参考：《山西医科大学》2002年硕士论文

【摘要】： 目的获得线粒体DNA HVI在太原地区汉族群体中分布的序列多态性数据，以探讨线粒体DNA序列多态性在法医学个体识别中应用的理论基础。并探索采用PCR-SSCP技术作为一种检测线粒体DNA序列多态性的筛选方法。方法随机抽取58名太原地区汉族群体无血缘关系个体的静脉血，EDTA抗凝，改良的TKM法提取mtDNA，PCR扩增、纯化后，应用377序列分析仪进行直接测序分析；对毛发、指甲、骨骼等微量或严重降解检材进行mtDNA序列分析；采集同一尸体血液、肌肉、肝、肾、心肌、不同部位的毛干等组织进行同一性检测；对20个两代家系进行突变观察。将上述58名无关个体的mtDNA扩增后，，10％非变性聚丙烯酰胺凝胶(含10％甘油)电泳，银染，进行SSCP分析。结果在线粒体DNAnt16081-16546区间发现有55种单倍型，其中53种是唯一的，另2种分别为2个和3个个体所共有。该区间共有74处碱基变异，其可变碱基的变异形式主要为碱基替换(转换和颠换)、插入和缺失；碱基转换(77.0％)明显高于颠换(9.46％)、插入(12.2％)、缺失(1.35％)。该区段基因变异度为99.8％，偶合概率为1.96％。微量血痕、毛发、指甲、骨骼进行山百医科大学 2002月卜祠吐士研究生毕习匕自仑文 mtDNA序列分析均能准确测定碱基序列。人体不同组织序列一致性检测中观察到头前部的一根毛发mtDNA序列16249处碱基N 为G/A，而其余组织此处碱基为A。家系突变观察结果显示一对母亲一孩子序列不一致，母亲为16193.ZC，孩子为16193.C。SSCP 分析结果在同一张凝胶上序列不同的8个样品，可平均检出5 种带型，检出率可达62.5%。结论所检测区间ntl6os一16546位于线粒体DNAHVI，从基因变异度和偶合概率两项指标，及mtONA可对那些ONA严重降解或不能提供足够核ONA的检材进行分析，显示mtDNA可作为良好的遗传标记进行个体识别。与不同人群比较结果中发现四个汉族群体间差异无显著性，而与瑞士高加索人群都存在显著性差异。人体不同组织一致性检验结果和家系突变中观察到异质型，异质型的出现并不会使mtDNA的法医学分析失效，但应考虑它对判定结果的影响。本文应用的荧光染料标记全自动测序技术具有准确性高，序列结果稳定;灵敏度高;电泳分离自动化等特点。但同时也存在一些不足，如测序需多次扩增，容易污染;该方法费用昂贵，需要特殊的仪器等。SSCP分析技术实验方法简单、易操作;实验成本低，可作为一种筛选大量嫌疑检材的方法。
[Abstract]:Objective to obtain the sequence polymorphism data of mitochondrial DNA HVI distribution in Taiyuan Han population and to explore the theoretical basis for the application of mitochondrial DNA polymorphism in forensic individual identification. PCR-SSCP was used as a screening method to detect mitochondrial DNA polymorphism. Methods in 58 unrelated individuals of Han nationality in Taiyuan area, the anticoagulant mtDNA-EDTA was extracted by modified TKM method. After purification, direct sequencing was performed by 377 sequence analyzers, and hair and fingernails were analyzed. Trace or severe degradation of bone samples were analyzed by mtDNA sequence, blood, muscle, liver, kidney, myocardium, hair stem of different parts of the same cadaver were collected for identity detection, and 20 generations of families were observed for mutation. After mtDNA amplification, 10% non-denatured polyacrylamide gel (including 10% glycerol) was detected by silver staining and SSCP analysis. Results there were 55 haplotypes in the mitochondrial DNAnt16081-16546 region, of which 53 were unique and the other two were shared by 2 and 3 individuals, respectively. There are 74 base variations in this region, and the variable bases are mainly in the form of base substitution (transformation and transversion, insertion and deletion; base conversion 77.0), which is obviously higher than that of 9.46%, with the insertion of 12. 2% and the absence of 1.35%. The genetic variation of this region is 99.8 and the probability of coupling is 1.96. Traces of blood, hair, nails, bones Shanbai Medical University In February 2002, the graduate student of Bushi Tutu graduated from Zilun. MtDNA sequence analysis can accurately determine the base sequence. The sequence of different tissues in human body is consistent. A mtDNA sequence of 16249 base N was observed in a hair in the anterior part of the head. G / A, and the rest of the tissues have a base A. Pedigree mutation observation showed a pair of Mother one child sequence is inconsistent, mother is 16193. ZC, child is 16193.C.SSCP Eight samples with different sequences on the same gel can be detected with an average of 5. 5% The detection rate was 62.5%. Conclusion the detected interval ntl6os-16546 is located in the mitochondrial DNA HVI. Genetic variability and coupling probability, and mtONA can be used to treat those ONA seriously. Degradation or failure to provide sufficient nuclear ONA samples for analysis, indicating that mtDNA can be used To identify individuals for good genetic markers. In comparison with different population groups, we find that There was no significant difference between Han population and Swiss Caucasian population. Difference. Heterogeneity was observed in the results of consistency test of different human tissues and family mutation. The appearance of heterogeneity does not invalidate the forensic analysis of mtDNA, but it should be considered Its effect on the result. Automatic sequencing technique for fluorescent dye labeling in this paper High accuracy, stable sequence results, high sensitivity; Electrophoretic separation automation, etc. Point. But at the same time there are some shortcomings, such as sequencing needs multiple amplification, easy to pollute; The method is expensive and requires special instruments and so on. The method of SSCP analysis is simple. Simple, easy to operate, low cost, can be used as a method to screen a large number of suspected materials.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2002
【分类号】：D919

【引证文献】