应用PCR-SSCP和PCR-RFLP方法分析线粒体DNA多态性及其法医学应用的研究

发布时间：2018-08-17 08:41

【摘要】： 前言国内外法医实验室采用的DNA遗传标记分为两大类:核基因组DNA和线粒体DNA(mtDNA)。与细胞核DNA相比,mtDNA有许多无法比拟的优点,如拷贝数高、对样木质量要求不高、对样木需求量低。这此特点非常适合用于细胞数量极少或严重降解的法庭科学生物样本,如骨骼、毛发、指甲、微量血痕等分析。因此在法医学领域,mtDNA的应用日益广泛。此外,除了满足法医物证学个体识别和亲缘关系鉴定这两项基本要求外,它已经越来越多的应用于法庭科学的各个方面。近年来,mtDNA的研究不断深入,线粒体DNA检出在很大程度上依赖检测方法的灵敏度,所以选择合适的分析技术就显得非常重要。就目前而言,在诸多的有关mtDNA的检测方法中,直接测定序列分析是使用最多、最广的方法。测序法要求有测序仪及相应的试剂盒,费用昂贵,操作费时,且对操作人员本身素质要求高,周期长,实际应用常受到限制,在基层难以开展。单链构象多态性(singlestrand conformation polymorphism,SSCP)和限制性片断长度多态性(Rrestrictionfragment length polymorphism,RFLP)技术是经典的突变筛选技术,突出特点是成本低,设备要求简单,周期短,非常适于实验室进行常规技术分型。鉴于mtDNA特有的环状结构和拷贝数多等特点,对降解和腐败有更强的抵抗力,在骨骼、毛发等特殊生物检材的法医学检验中发挥了重要作用,为了建立一种适合于基层实验室分析的技术,本研究将利用PCR-SSCP和PCR-RFLP方法进行线粒体DNA多态性进行分析,为法医学个人识别、亲权鉴定及群体遗传学提供基础数据。材料和方法 1、150例辽宁汉族健康无关个体及30例两代家系血液样品200μl、5例死后12h的各主要脏器(心、肝、脾、肺、肾、脑)0.5cm×0.5cm×0.5cm和5例甲醛固定石蜡包埋的组织切片(6μm厚)均由中国医科大学法医学院提供。 2、利用primer5.0设计引物,进行PCR扩增。通过PCR-SSCP和PCR-RFLP方法分析线粒体DNA多态性。利用两种技术分别对150例辽宁汉族健康无关个体进行分析,获得单倍型频率,并进行组织一致性检验以及在亲权鉴定和降解检材的法医学应用。结果 1、用PCR-SSCP法获得样品清晰的单倍型图谱,150份无关个体血样检出9种单倍型,DP值分别为0.8809。在150例随机个体中,RsaI和MnlI酶切分别发现3和8种表型,DP值分别为0.1073,0.6698。联合两种酶切,发现12种表型,DP值为0.7078。应用两种技术对150例随机个体发现33种组合单倍型,DP值为0.925。 2、在5例正常组织中,各个组织的RFLP带型和SSCP带型完全相同。 3、在30个家系中,母子的RFLP带型和SSCP带型完全相同。 4、5例甲醛固定包埋组织的RFLP分型和SSCP分型与对照组织一致。结论 1、PCR-RFLP和PCR-SSCP方法用于法医物证的初步筛查简便、快速、经济,适合作为基层实验室线粒体DNA分析的常规方法。 2、mtDNAHVI16106-16297区域在中国辽宁地区汉族具有良好的多态性分布,在法医学上具有较高的应用价值。 3、线粒体DNA多态性较常规STR分析更适合于甲醛固定石蜡包埋组织等非常规法医物证检材的DNA多态性分析。
[Abstract]:Preface
DNA genetic markers used in forensic laboratories at home and abroad can be divided into two categories: nuclear genomic DNA and mitochondrial DNA (mtDNA). Compared with nuclear DNA, mtDNA has many incomparable advantages, such as high copy number, low requirement for sample wood quality and low demand for sample wood. This feature is very suitable for courts with very few cells or serious degradation. Scientific biological samples, such as bone, hair, nails, trace bloodstains and so on, have been widely used in forensic science. In addition, besides satisfying the two basic requirements of forensic biology, such as individual identification and kinship identification, mtDNA has been more and more applied in all aspects of forensic science.
In recent years, the research of mtDNA has been deepening. The detection of mitochondrial DNA depends on the sensitivity of detection methods to a great extent, so it is very important to select the appropriate analysis technology. The instrument and its corresponding kits are expensive, time-consuming, and require high quality of the operators themselves. The practical application is often limited and difficult to carry out at the grass-roots level. Single strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) The technology is a classical mutation screening technology, which is characterized by low cost, simple equipment requirements, short cycle, and very suitable for laboratory routine technical typing.
In view of the characteristics of mtDNA, such as its unique ring structure and more copies, it has a stronger resistance to degradation and corruption, and plays an important role in forensic testing of bone, hair and other special biological samples. In order to establish a suitable technology for basic-level laboratory analysis, this study will use PCR-SSCP and PCR-RFLP methods to carry out more mitochondrial DNA. Analysis of the state provides basic data for forensic personal identification, paternity testing and population genetics.
Materials and methods
The blood samples of 1,150 unrelated healthy individuals and 30 families of two generations in Liaoning Province were 200 ml. The major organs (heart, liver, spleen, lung, kidney, brain) 0.5 cm *0.5 cm *0.5 cm and 5 formaldehyde-fixed paraffin-embedded tissue sections (6 micron thick) were provided by the Forensic Medical College of China Medical University 12 hours after death.
2. Primer 5.0 was used to design primers for PCR amplification. Mitochondrial DNA polymorphisms were analyzed by PCR-SSCP and PCR-RFLP. The haplotype frequencies of 150 healthy unrelated individuals in Liaoning Han nationality were analyzed by two techniques, and the tissue consistency test was carried out.
Result
1. A clear haplotype map was obtained by PCR-SSCP. Nine haplotypes were detected in 150 unrelated blood samples with DP values of 0.8809. In 150 randomized individuals, 3 and 8 phenotypes were detected by RsaI and MLI digestion, respectively, with DP values of 0.1073 and 0.6698. Twelve phenotypes were found with DP values of 0.7078. 33 combinations of haplotypes were found in individuals, with a DP value of 0.925..
2, in 5 normal tissues, the RFLP band patterns and SSCP band patterns of all tissues were identical.
3, in the 30 families, the RFLP band pattern and the SSCP band pattern of mother and child are the same.
The RFLP typing and SSCP typing of formaldehyde fixed embedded tissues in 4,5 were consistent with those in control tissues.
conclusion
1. PCR-RFLP and PCR-SSCP are simple, rapid and economical methods for preliminary screening of forensic physical evidence. They are suitable for routine analysis of mitochondrial DNA in primary laboratories.
2. The mtDNA HVI16106-16297 region has a good polymorphism distribution in the Han nationality in Liaoning Province of China, and has a high application value in forensic medicine.
3. Mitochondrial DNA polymorphism is more suitable for DNA polymorphism analysis of unconventional forensic material such as formaldehyde fixed paraffin embedded tissue than conventional STR analysis.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

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