三个miniSTR基因座荧光标记复合扩增体系的建立及其法医学应用研究

发布时间：2018-04-13 04:16

本文选题：法医遗传学 + miniSTR　；参考：《四川大学》2007年硕士论文

【摘要】： 目的本课题旨在建立新的miniSTR荧光标记复合扩增体系，并按照美国DNA分析方法技术工作组(TWGDAM)的指导方案，进行法医学应用性研究。方法从已有国内频率资料的STR基因座中筛选出可用于设计MiniSTR引物的基因座，进行引物设计；用未标记的新引物在银染显色方法下进行复合扩增实验，选出复合成功基因座定购荧光标记引物；制作等位基因分型标准物；采用美国ABI310遗传分析仪进行荧光标记引物复合扩增试验，并优化扩增条件，对不同退火温度下复合扩增的效果进行研究；对复合扩增系统的灵敏度、基因座的种属特异性、案例应用、降解模型、陈旧样本等法医学应用进行研究。结果成功设计了D19S591，D2S2944，D18S872基因座的小于130bp的miniSTR扩增引物，成功建立了D19S591，D2S2944，D18S872的miniSTR荧光标记复合扩增体系。这一扩增体系条件易于优化，采用国产Taq酶进行扩增，即可得到满意的扩增产物和分型结果。相对于国外昂贵的试剂盒，它是一种成本低廉但效率高的STR基因座复合扩增体系。法医学应用性研究表明：三个基因座有较高的种属特异性；该体系的检测灵敏度为0．25ng模板DNA；通过6个实际检案证明，该体系能用于法医学个人识别和亲权鉴定；通过对陈旧样本和降解模型的研究证明，该体系对降解检材有良好的扩增效果，较常规大片段试剂盒扩增成功率高，可用于常规试剂盒扩增失败的降解样本的分型。结论D19S591，D2S2944，D185872的miniSTR荧光标记复合扩增体系可应用ABI-310检测平台，获得可靠的遗传学数据。该体系扩增条件易于优化，成本低廉，分型结果稳定，种属特异性好，灵敏度和准确度高，，对降解检材扩增成功率高，可以应用于法医学实际检案，特别适合对陈旧样本和降解检材的法医学检测。
[Abstract]:Objective to establish a new miniSTR fluorescent labeling multiplex amplification system, and to carry out forensic application research according to the guidance of the DNA analysis technique working group in the United States.Methods STR loci which could be used to design MiniSTR primers were screened from STR loci with existing domestic frequency data, and the primers were designed, and the multiplex amplification experiments were carried out with new unlabeled primers by silver staining method.The fluorescent marker primers were selected for complex successful loci, the allelic genotyping standard materials were made, and the multiplex amplification test of fluorescent marker primers was carried out by ABI310 genetic analyzer in the United States, and the amplification conditions were optimized.The effects of multiplex amplification at different annealing temperatures were studied, and the sensitivity of multiplex amplification system, gene locus specificity, case application, degradation model, old samples and other forensic applications were studied.Results the miniSTR primers of D19S591mD2S2944nD18S872 locus were designed successfully, and the multiplex amplification system of D19S591D2S294nD18S872 was successfully established.The conditions of this amplification system are easy to be optimized, and satisfactory amplification products and typing results can be obtained by using domestic Taq enzyme.Compared with foreign expensive kit, it is a low cost and high efficiency STR locus multiplex amplification system.Forensic application studies showed that the three loci had high species-specificity, the sensitivity of the system was 0.25ng template DNA, and the system could be used for forensic personal identification and paternity identification.The study on the old samples and degradation models showed that the system had a good amplification effect on the degradation samples, and had a higher success rate than the conventional large fragment kits, and could be used for the typing of degraded samples which failed in the amplification of the conventional kits.Conclusion the miniSTR multiplex amplification system of D19S591D2S2944 and D185872 can be used for ABI-310 detection to obtain reliable genetic data.The system is easy to optimize, low cost, stable typing results, good species specificity, high sensitivity and accuracy, high success rate of amplification of degradable samples, and can be used in forensic medical practice.It is especially suitable for forensic examination of old samples and biodegradable samples.
【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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