STR基因座嵌合引物复合扩增及其法医学应用研究

发布时间：2018-07-21 09:59

【摘要】： 目的本课题旨在探索一种新的STR基因座复合扩增方法，我们称为嵌合引物STR复合扩增法。应用荧光标记毛细管电泳激光自动检测技术平台，建立一套新的法医STR基因座复合扩增体系，并按照美国DNA分析方法技术工作组(The Technology Working Group on DNA Analysis Methods，TWGDAM)的指导方案进行法医学实用性研究。方法利用PCR嵌合引物复合扩增技术扩增D1S1612、D9S2026、D20S161、D6S477 STR基因座，用聚丙烯酰胺电泳对PCR产物分离，银染；应用毛细管电泳激光自动分析系统对荧光标记的嵌合引物复合扩增产物进行检测；用混合样本、不同的组织、不同的介质、常见的动物在不同的模板量、不同的温度条件下对嵌合引物复合扩增系统进行测试，用实际案例进行验证。结果通过采用公共引物和基因座特异性嵌合引物的方法，，建立了法医STR基因座嵌合引物复合扩增方法。这种方法扩增条件易于优化，不同基因座扩增条件的齐同性要求不高，对引物二聚体有一定程度的包容性，可用国产Taq酶做出四川大学博士学位论文董建国稳定而满意的结果，是一种成本低效率高的法医STR基因座复合扩增的方法。建立了DIS1612、D952026、D205161、D65477 STR 基因座荧光标一记嵌合引物复合扩增毛细管电泳激光自动分析体系，同时证明嵌合引物复合扩增方法与毛细管电泳激光自动分析方法相兼容。按照TWGDAM指导方案的基本要求，对该体系进行了法医实用性研究。证明该体系对同一个体的不同组织检测结果一致;对常见介质上的检材及混合样本具有检测能力;对80OC 放置6天的检材、户外阳光下放置14天的检材仍然可分型;可对 0.25ng的微量DNA模板分型;与15种常见动物和微生物无交叉反应;应用于案例检测，与鉴定结论一致。结论建立了法医四个遗传标记DIS1612、D952026、 D205161、D65477新的荧光标记嵌合引物复合扩增体系，证明该体系可成功应用于荧光标记毛细管电泳激光自动分析检测平台。法医应用性研究证明，新的荧光标记嵌合引物复合扩增体系分型结果稳定，重复性好，灵敏度高，对陈旧斑痕和混合斑具有检测能力，能够对常见介质上的斑痕正确分型，与常见的种动物及微生物无交叉反应，可成功应用于法医检案。
[Abstract]:Objective to explore a new method for multiplex amplification of STR loci, which is called chimeric primer STR multiplex amplification. A new multiplex amplification system of forensic STR loci was established by using fluorescence labeled capillary electrophoresis laser automatic detection platform. The practicability of forensic science was studied according to the guidance of the Technology working Group on DNA Analysis methods (TWGDAM). Methods D1S1612D9S2026 D20S161D6S477 STR loci were amplified by PCR chimeric primers. PCR products were isolated by polyacrylamide electrophoresis and stained with silver. The fluorescent labeled chimeric primer compound amplification products were detected by capillary electrophoresis laser automatic analysis system, mixed samples, different tissues, different media, common animals in different templates, The chimeric primer compound amplification system was tested under different temperature conditions and verified by practical cases. Results by using common primer and locus specific chimeric primer, a multiplex amplification method of forensic STR locus chimeric primer was established. The amplification conditions of this method are easy to optimize, the homogeneity of different loci amplification conditions is not high, and the primer dimer has a certain degree of inclusiveness. Using domestic Taq enzyme to make Sichuan University Dong Jianguo's stable and satisfactory results in his doctoral thesis, It is a low cost and high efficiency method for multiplex amplification of forensic STR loci. DIS1612 D952026 (D205161) D65477 STR locus fluorescent marker and chimeric primers were used to amplify capillary electrophoresis (CE) laser automatic analysis system, and the results showed that DIS1612, D952026 and D205161D65477 STR locus were amplified by capillary electrophoresis. At the same time, it was proved that the multiplex amplification method of chimeric primers was compatible with the laser automatic analysis method of capillary electrophoresis. In accordance with the basic requirements of the TWGDAM guidance scheme, the forensic practicability of the system was studied. It is proved that the system has the same ability to detect fruit formation in different tissues of the same individual, and has the ability to detect samples and mixed samples from common media. The samples placed in 80OC for 6 days and those stored in outdoor sunlight for 14 days could still be classified. MicroDNA template typing of 0.25ng; no cross reaction with 15 common animals and microbes. It is consistent with the conclusion of identification. Conclusion the multiplex amplification system of four genetic markers, DIS1612, D952026, D205161, D65477, was established. It is proved that the system can be successfully applied to the automatic detection platform of fluorescence labeled capillary electrophoresis with laser. The results of the new fluorescent marker chimeric primer multiplex amplification system were stable, reproducible and sensitive. It has the ability to detect the old marks and mixed spots, and can correctly type the marks on the common media. No cross-reaction with common animals and microorganisms can be successfully used in forensic examination.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2003
【分类号】：D919

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