辽宁地区汉族人群D17S518基因座多态性及其毛发检材的应用研究

发布时间：2018-02-05 05:53

  本文关键词： 二核苷酸简单串联重复顺序 D17S518 PCR-STR 毛发检材 辽宁汉族　出处：《中国医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 前言 短串联重复序列(short tandem repeats,STR)又称微卫星DNA(mierosatellite),是指重复单位只有1～6bp、重复次数一般在数次至几十次之间的串联重复DNA序列。STR在人类基因组中分布广泛、数量较多、等位基因片段长度较短、扩增成功率高,是目前法医物证鉴定中应用最广泛的DNA长度多态性遗传标记。 然而,对于毛发等富含角化细胞的特殊法医物证检材,由于其细胞核发生明显的转移,核基因组DNA含量少且分解严重,常规方法检测法医物证鉴定中最常用的四核苷酸STR基因座时,常难以获得满意的结果。线粒体DNA(mitochondrialDNA,mtDNA)检测作为法医学应用序列多态性分析进行个人识别的典型实例,在毛发等非常规检材的鉴定中具有重要的实用价值。但mtDNA特殊的遗传特征、突变率较高、信息量较低、检测技术复杂等因素的影响,在一定程度上限制了mtDNA的法医学应用。因此,选择多态性程度较高且等位基因片段更短,适用于毛发等检材DNA多态性分析的遗传标记,是法医物证工作者共同关心的课题。 D17S518基因座位于人类第17号染色体上,是由(CA)n核心序列构成的二核苷酸简单串联重复序列。本研究首先应用PCR扩增结合聚丙烯酰胺凝胶电泳分离的方法,调查D17S518基因座在辽宁汉族人群中的频率分布,以评估该遗传标记的法医学应用价值,并对其应用于毛发检材的可行性及影响因素进行了初步研究,为该基因座在更广泛领域的应用奠定了基础。 材料与方法 根据文献合成特异性扩增D17S518基因座的引物,应用PCR技术和聚丙烯酰胺凝胶电泳分离结合银染显谱的方法,对120例中国辽宁地区无血缘关系的汉族个体血液样本进行D17S518基因座分型,并通过DNA序列分析确定其等位基因;对采用不同方法(酚/氯仿法和Chelex—100法)提取DNA,不同数量(1根、2根和5根)和不同区段(远段、中段和近段)毛发检材的D17S518基因座扩增结果进行比较。 结果 在120例中国辽宁地区无血缘关系汉族个体中,检出D17S518基因座的5种等位基因及12个基因型,根据DNA序列分析结果将扩增片段长度为88bp、90bp、94 bp、98 bp、100 bp的5种等位基因分别命名为D17S518*15、D17S518*16、D17S518*18、D17S518*20和D17S518*21。12种基因型及频率分别为:15/15为12例(10%),16/16为13例(10.7%),18/1 8为3例(2.5%),15/16为21例(17.5%),15/18为23例(19.2%),15/20为6例(5%),15/21为2例(1.7%),16/18为20例(16.7%),16/20为8例(6.7%),16/21为5例(4.2%),18/20为4例(3.3%),18/21为3例(2.5%);5种等位基因频率分别为:D17S518*15=0.317、D17S518*16=0.333、D17S518*18=0.233、D17S518*20=0.075、D17S518*21=0.042。应用Powerstatsvl2软件对D17S518基因座群体调查结果进行数据分析,获得如下法医学应用参数:杂合度(H)为0.767,个人识别几率(DP)为0.872,偶合几率(Pm)为0.128,非父排除率(PE)为0.539,多态信息含量(PIC)为0.68。 6个个体毛发检材的D17S518基因座扩增产物检测结果:①采用酚/氯仿方法提取模板DNA,其扩增产物检出率随毛发数量的增加而提高,即1根为2/6、2根为4/6、5根为5/6;而采用Chelex-100法提取模板DNA的毛发检材均未检出扩增产物。②同一毛发不同区段的扩增产物检出结果不同;不同毛发中、远段部分扩增产物检出规律不同,但近段毛发均可检出扩增产物。 结论 1、D17S518基因座在中国辽宁地区汉族群体中具有良好的多态性分布,在法医学上具有较高的应用价值。 2、二核苷酸简单串联重复序列的STR基因座适用于毛发等非常规法医物证检材的DNA多态性分析。 3、酚/氯仿法提取毛干DNA的扩增效果明显优于Chelex-100法。
[Abstract]:Preface
Short tandem repeat (short tandem, repeats, STR) and microsatellite DNA (mierosatellite), refers to the repeat unit is only 1 ~ 6BP repeat number in general repeat DNA.STR is widely distributed in the human genome, several to dozens of times between the larger number of allele fragment length is short, amplification was successful. High rate, is currently the most widely used in forensic evidence identification DNA length polymorphism genetic markers.
However, for the special forensic samples such as hair in keratinocytes, because of its obvious nuclear transfer, nuclear DNA content and less serious decomposition, conventional methods of forensic identification of tetranucleotide STR loci are most commonly used, often is difficult to obtain satisfactory results. The mitochondrial DNA (mitochondrialDNA, mtDNA) detection as a forensic application of sequence polymorphism analysis of typical examples of personal identification, it has important practical value in the identification of hair and other non conventional materials. But the genetic characteristics of special mtDNA, the mutation rate is higher, the amount of information is low, influence of complex detection technology and other factors, to some extent limit the forensic mtDNA application. Therefore, choose a higher degree of polymorphism and allele for genetic markers in shorter hair samples DNA polymorphism analysis, forensic evidence is the common concern Subject.
D17S518 gene is located on human chromosome seventeenth is composed of (CA) n core sequence consisting of the dinucleotide. In this study we used PCR amplification combined with separation method of polyacrylamide gel electrophoresis, the investigation of D17S518 loci in Liaoning Han population in the frequency distribution, used to assess the genetic markers in forensic. And the feasibility and impact factors of its application in hair samples was studied, which laid the foundation for the application of this locus in a wide range of areas.
Materials and methods
The synthesis of specific primers according to the amplification of the D17S518 locus, separation and silver staining significantly spectrum using PCR and polyacrylamide gel electrophoresis in 120 cases of China unrelated Liaoning Han individual blood samples were D17S518 genotype, and determine its allele by DNA sequence analysis of different methods; (phenol / chloroform method and Chelex 100 method) to extract DNA, different number (1, 2 and 5) and different sections (distal, middle and proximal) D17S518 loci in hair samples were compared.
Result
鍦，

本文编号：1492314