【作者】 邓建强；

【导师】 侯一平；

【作者基本信息】 四川大学 ， 法医学， 2004， 博士

【摘要】 目的:本文拟对简并寡核苷酸引物PCR(DOP)和扩增前引物延伸(PEP)两种全基因组扩增技术在法医学实践中的应用价值进行探讨,对其应用于法医学实践的可行性进行系统评价。 方法:对两种全基因组扩增(WGA)技术中可能出现STR滑脱现象的影响因素进行研究,然后在控制这些滑脱影响因素的基础上建立稳定可靠的技术方案;将该技术方案应用于法医学的单基因座扩增聚丙烯酰胺凝胶电泳银染检测系统、多色荧光标记复合扩增毛细管电泳激光自动检测系统以检验两种技术应用于法医学常用STR基因座的可靠性;将该技术应用于法医学常见微量检材单根毛发和指纹的DNA分析,对其法医学应用价值进行评估。 结果:初步建立了基于DOP技术的STR滑脱机制模型,对WGA技术中可能导致STR滑脱现象的影响因素得到了解和阐述;在控制WGA技术中可能导致滑脱现象发生的影响因素的基础上,通过优化组合,建立了可靠稳定的PEP和DOP两种WGA技术方案;两种技术方案应用于法医学单基因座STR分析和STR复合扩增分析,均获得了满意的分析结果;建立了通过显微操作捕获细胞进行准更多还原

【Abstract】 Objective : Genetic analysis from forensic microsamples is a urgent, difficult task in forensic science, because it is frequently limited by the amount of specimen available in forensic practice. Much effort has been carried out to resolve this difficulty. Whole genome amplification (WGA) technology has been thought to be a powerful, reliable and efficient strategy in analysis of minute amount DNA on many fields, for it can generate large quantity of DNA from minute DNA templates with unbiased way and its products can theoretically cover almost all the regions of genome. So, we employed two most used WGA methods-primer extension preamplification (PEP) and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) into forensic DNA analysis, and made a systemic study to evaluate its application in forensic science.Methods: We launch our study with the objective to get STRgenotyping result without STR slippage and no-specific products. We first observed factor that might result in STR slippage, and with these knowledge established standard and steady PEP and DOP approaches by optimizing several factors, then these approaches were used in single STR, multiplex STR loci, single shed hair and fingerprint DNA analysis for forensic purpose.Result: We found some factors that may result in STR slippage and even built up a STR slippage model by controlling several factors. The PEP and DOP approaches were established by optimizing several factors and proved to be reliable, unbiased, steady, sensitive, and efficient in forensic single STR analysis, multiplex STR analysis and single shed hair analysis. However, we did not succeed in STR genotyping of fingerprint sample, which may result from the DNA extracting method we used not sensitive enough. In silver detecting system, PEP and DOP methods can analys the quantity of DNA template at least 0.125ng in all samples, while the least needed quantity of DNA template in general PCR is 0.5ng. We also established an accurate, reliable method to get single cell through micromanipulation for DNA sensitive analysis and used it in our study. In multiplex PCR-ABI310 detecting system, PEP and DOP methods can succeed in genotyping Amelogenin and 9 loci in all studied samples at least 5 cells, while general PCR needed more than 10 cells.Conclusion: Approaches we have established in this study were reliable, steady enough for forensic purpose and can be used in forensic practice. The micromanipulation approaches we establishedhere was reliable and accurate method for DNA sensitive analysis. The PEP and DOP methods not only were more sensitive than general PCR, but also can produce large quantity DNA from microsample DNA, which can meet the need of both re-typing and check-typing for forensic purpose.更多还原