D3S3053，D6S474，D20S482，D1GATA113，D2S1776，D4S2408 miniSTR基因座四

发布时间：2018-06-22 06:41

本文选题：miniSTR + DNA分型　；参考：《河北医科大学》2008年硕士论文

【摘要】： 目的:自上世纪80年代以来,短串联重复序列(short tandem repeats, STR)遗传标记系统,由于在人类基因组中分布广泛,高度的遗传多态性以及简单快捷的检测方法而成为法庭科学鉴定中最常用、发展最成熟的技术,被广泛应用于法医学等领域,STR数据成为各国法庭科学数据库的主要构成。目前法医学主要运用常染色体STR基因座和线粒体DNA遗传标记,能够解决大部分个体识别和亲权鉴定案例。然而,在许多法医案例中,DNA样本由于各种原因而被高度降解或被混入环境污染物,在使用商品化STR复合扩增试剂盒进行检测时,片段较长的STR基因座往往无法进行有效的扩增,片段检测不到信号,而这个问题随着PCR反应产生大量的PCR产物而被扩大。miniSTR基因座分析技术在设计引物时,使其尽可能靠近核心重复序列而缩短扩增片段长度,用于高度降解的DNA样本检测可提高检测成功率。miniSTR基因座被欧洲DNA分型工作组(the european DNA profiling group, EDNAP)定义为继STR之后的新一代遗传标记。美国、日本、西班牙、新加坡、韩国等国家也相继报道了miniSTR基因座的群体遗传学数据及其法医学应用价值,而我国相关报道较少并缺乏群体遗传学数据。本研究选取D3S3053, D6S474, D20S482, D1GATA113, D2S1776, D4S2408等六个miniSTR基因座,调查其在中国汉族人群中的遗传多态性,并探讨其法医学应用价值。方法:采用天根血液基因组DNA提取试剂盒提取120份河北汉族无关健康个体全血基因组DNA。将D20S482、D2S1776两个基因座的上游引物5’端用6-FAM荧光标记,D3S3053、D1GATA113两个基因座的上游引物5’端用HEX荧光标记,D6S474、D4S2408两个基因座的上游引物5’端用TAMRA荧光标记,对所有样本的六个miniSTR基因座进行PCR复合扩增,其中D3S3053、D6S474、D20S482三个基因座进行复合扩增,D1GATA113、D2S1776、D4S2408三个基因座进行复合扩增。复合扩增反应体系为10μl,分别加入各个基因座特异性的引物,并设定适宜的退火温度,经28个循环后,可得到各个基因座长度不等的等位基因片段。PCR复合扩增产物在ABI PRISM 310基因分析仪上自动完成进样电泳和电泳结果数据收集,结果用Genemapper 3.2计算扩增产物片段相对大小并进行样本基因型分型。计算各基因座基因频率及各法医学参数。选取本法医鉴定中心既往亲子鉴定的家系样本9例,对6个miniSTR基因座进行遗传稳定性的研究。提取同一尸体的心脏、肝脏、脾脏、肺组织、肾脏、脑组织、肌肉组织、皮肤及尸体血痕检材进行组织同一性分析。将9947A DNA样本稀释成1ng、0.5ng、0.1ng、0.05ng进行灵敏度检测。此外,将新鲜血液放置于夏季的自然环境中一周、两周、四周和八周模拟降解检材,应用本研究构建的6个miniSTR基因座荧光标记复合扩增分型体系及商品化常规STR试剂盒进行DNA分型,比较二者分型的成功率。结果:本研究建立了两组荧光复合扩增检测6个miniSTR基因座基因型的方法。复合扩增检测样本的分型结果显示,六个miniSTR基因座等位基因都获得了清晰的基因型分型结果,无干扰分型的非特异性扩增产物,对6个基因座都可实现准确分型。相关遗传学参数:六个基因座的扩增片段长度均小于150bp。120名河北汉族无关个体中D3S3053、D6S474、D20S482、D1GATA113、D2S1776、D4S2408基因座分别检出6、6、7、8、8、7个等位基因和14、16、15、27、27、23种基因型,基因型频率分布经χ2检验均符合Hardy-Weinberg平衡(P0.05)。六个基因座在中国汉族人群的杂合度观察值(Ho)分别为0.652,0.758,0.758,0.667,0.617,0.85;多态性信息含量(PIC)分别依次为0.64,0.68,0.69,0.83,0.85,0.83;非父排除率(PE)分别依次为0.298,0.523,0.523,0.313,0.271,0.662;个人识别力(PD)分别依次为0.862,0.857,0.877,0.943,0.947,0.917。六个miniSTR基因座的累积非父排除概率为0.97,累积个人识别力为0.9999994。遗传稳定性分析:对9个已经确定亲权关系的家系样本研究表明,父母的等位基因能够稳定地遗传给子女,符合孟德尔遗传定律。组织同一性分析:对来自同一尸体不同组织的检测表明,六个基因座在同一个体中具有同一性,符合STR基因座用做法医DNA分析的标准。系统灵敏度研究:在DNA含量为0.1ng时, Identifile试剂盒扩增产物产量比较低,不易进行正确的分型,而应用miniSTR复合扩增系统对0.05ng的DNA还可以进行分型。降解微量检材研究:对降解两个月的检材,虽然扩增的特异度有所下降,但对分型结果没有干扰,在六个miniSTR基因座中均得到了完整分型,显示了miniSTR技术比常规STR试剂盒具有更高的分型成功率。结论:D3S3053、D6S474、D20S482、D1GATA113、D2S1776和D4S2408六个miniSTR基因座在河北地区汉族群体中具有较好的遗传多态性,符合孟德尔遗传规律,具有组织同一性。本研究建立的两组荧光标记复合扩增体系分型结果准确、稳定可靠,灵敏度达0.05ng,对于分析微量、降解DNA的成功率较常规STR试剂盒明显升高,可用于法医学亲权鉴定与个人识别,并在高度降解检材的检测中具有较高的应用价值,而且可作为常规STR试剂盒的有益补充,提高系统效能。
[Abstract]:Objective: since 80s, the short tandem repeats (STR) genetic marker system has been widely used in forensic science and other fields because of its wide distribution, high genetic polymorphism and simple and fast detection methods in the human genome. STR data has become the main component of the National Forensic Science Database. Currently, forensic medicine mainly uses the autosomal STR loci and mitochondrial DNA genetic markers to solve most individual identification and paternity cases. However, in many forensic cases, DNA samples are highly degraded or mixed into environmental pollutants for a variety of reasons. When the commercialized STR complex amplification kit was used to detect the longer STR loci, the fragment was often unable to be amplified effectively, and the fragment was not detected. The problem was enlarged with the PCR reaction to produce a large number of PCR products, and the.MiniSTR gene pedestal analysis technique was expanded as close as possible to the core repeat sequence when the primers were designed. Shortening the length of the amplified fragment, for highly degraded DNA samples, can improve the detection success rate of the.MiniSTR loci by the European DNA typing group (the European DNA profiling group, EDNAP) as a new generation of genetic markers following STR. The United States, Japan, Spain, Singapore, and South Korea have also reported the miniSTR gene. The population genetics data and the application value of forensic medicine are less and lack of population genetics data in our country. This study selected six miniSTR loci, such as D3S3053, D6S474, D20S482, D1GATA113, D2S1776, D4S2408 and so on, to investigate the genetic polymorphism of the Chinese Han population and to explore the application value of their forensic medicine.
Methods: the genomic DNA extraction kit was used to extract 120 copies of the whole blood genome DNA. of unrelated healthy individuals from Hebei Han. The upstream primers of D20S482, D2S1776 two loci were labeled with 6-FAM fluorescence, D3S3053, D1GATA113 two loci were labeled with HEX fluorescence, D6S474, D4S2408 two loci. The 5 'end of the primer was labeled with TAMRA fluorescence, and the six miniSTR loci of all the samples were amplified by PCR, in which three loci of D3S3053, D6S474, D20S482 were amplified, and three loci of D1GATA113, D2S1776 and D4S2408 were multiplex amplified. The compound amplification reaction body was 10 micron L, and the specific primers were added to each loci, respectively. And the suitable annealing temperature was set. After 28 cycles, the.PCR composite amplified products of different allele length could be obtained on the ABI PRISM 310 gene analyzer. The results were obtained by using Genemapper 3.2 to calculate the relative size of the amplified product fragments and carry out the sample genotypes. The genetic stability of the 6 miniSTR loci was studied in 9 family samples of the previous paternity test in the forensic identification center. The samples of the same body were extracted from the heart, liver, spleen, lung tissue, kidney, brain tissue, muscle tissue, skin and corpse blood stains. 9947A DNA samples were diluted into 1ng, 0.5ng, 0.1ng, 0.05ng for sensitivity detection. In addition, the fresh blood was placed in the summer natural environment for a week, two weeks, four weeks and eight weeks, and 6 miniSTR loci fluorescent tagged composite amplification systems and commercial conventional STR reagents were constructed. The box is typed by DNA, and the success rate of the two types is compared.
Results: two groups of fluorescent multiplex amplification were established to detect the genotypes of 6 miniSTR loci. The typing results showed that six miniSTR loci alleles obtained clear genotyping results without interference type non specific amplification products, and all 6 loci could be accurately divided. Related genetic parameters: the length of the amplified fragment of the six loci was less than the D3S3053, D6S474, D20S482, D1GATA113, D2S1776, and D4S2408 loci in the unrelated individuals of the Hebei Han people, and the D4S2408 loci detected the 6,6,7,8,8,7 alleles and the 14,16,15,27,27,23 genotypes respectively. The frequency distribution of the basic genotype was all in line with the Hardy-Weinberg level by the x 2 test. P0.05. The observation value of heterozygosity (Ho) of the six loci in Chinese Han population (Ho) was 0.652,0.758,0.758,0.667,0.617,0.85, and the polymorphism information content (PIC) was respectively 0.64,0.68,0.69,0.83,0.85,0.83, and the non parent exclusion rate (PE) was respectively 0.298,0.523,0.523,0.313,0.271,0.662, and the individual recognition power (PD) in turn was 0.862,0., respectively. The cumulative non paternal exclusion probability of 857,0.877,0.943,0.947,0.917. six miniSTR loci is 0.97, and the cumulative individual recognition power is 0.9999994. genetic stability analysis. The family sample study of 9 identified parental relationships shows that the parents' alleles can be inherited stably to the daughter and conform to Mendel's law of inheritance. Analysis: the detection of different tissues from the same body showed that the six loci had the same character in the same individual, which accords with the standard of the STR loci for forensic DNA analysis. System sensitivity study: when the DNA content is 0.1ng, the production of Identifile kit is low, and the correct typing is not easy, and the miniSTR compound expansion is used. The increasing system can also be classified for the DNA of 0.05ng. Degradation of trace material for two months degradation, although the specificity of the amplification has declined, but there is no interference to the results of the classification, and the complete typing in the six miniSTR loci shows that the miniSTR technology has a higher classification success rate than the conventional STR kit.
Conclusion: the six miniSTR loci of D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408 have good genetic polymorphisms in the Han population in Hebei region, which conform to the genetic regularity of Mendel and have the identity of tissue. The results of the two groups of fluorescent labeled compound amplification in this study are accurate, stable and reliable, and the sensitivity is 0.05ng, and In the analysis of trace amounts, the success rate of DNA degradation is significantly higher than that of the conventional STR kit. It can be used in forensic paternity identification and personal identification, and has high application value in the detection of highly degraded materials, and can be used as a useful supplement to the conventional STR kit and improve the efficiency of the system.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919

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