【作者】 董建国；

【导师】 侯一平；

【作者基本信息】 四川大学 ， 法医遗传学， 2003， 博士

【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。更多还原

【Abstract】 Objective. The short tandem repeat (STR) loci in human genome are important markers for Forensic DNA typing. The purposes of our work are to establish a simplified method of multiplex PCR based on chimeric primers for STR loci, to develop a set of fluorescent quadriplex STR system for forensic DNA typing based on this method, and to validate the forensic application of the system under the guidelines of TMGDAM (The Technology Working Group on DNA Analysis Methods ) in order to address concerns presented in today’s legal environment. Methods. A set of chimeric primers and universal primers were designed by us to carry out a multiplex PCR for four STR loci. Using our primer set, four STR loci, D1S1612, D9S2026, D20S161 and D6S477 were amplified and detected by polyacrylamide gel electrophoresis and gels were sliverstained. Also,these STR loci were amplified by multiplex PCR using our primer set, but the universal primers were fluorescent-labeled. The products of the multiplex PCR were genotyped by ABI PRISM 310 Genetic Analyzer. The quadriplex STR systems were evaluated under varies conditions. Validation study of Forensic Science included concordance studies, sensitivity, reproducibility, and species specificity, as well as performance of typing forensic samples. Result. A method of multiplex PCR based on both chimeric and universal primers was established. A fluorescent quadriplex STR system, including D1S1612, D9S2026, D20S161 and D6S477, was developed on the basis of the multiplex PCR. The reaction conditions of multiplex PCR were shown to be optimal yet robust enough to withstand moderate variations. Genotyping with 0.25 ng was successful. Alleles at four STR loci could be detected in mixture sample with a proportion of 1 to 10. Environmental and matrix source analyses revealed an ability of our fluorescent quadriplex STR system to obtain complete genotypes in all samples except those exposed to 80癈 for 12-48 days. Conclusion. The multiplex PCR based on both chimeric and universal primers is an efficient method to amplify STR loci. It is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. The use of chimeric primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR. Our fluorescent quadriplex STR system can be analyzed by capillary electrophoresis and provide a newsources of typing regent for this technique platform with laser detecting system. The validation study showed that our fluorescent quadriplex STR system satisfied the TMGDAM guideline and it can be used for forensic casework.更多还原