6个miniSTTR基因座四色荧光分型体系的构建及法医学应用

发布时间：2018-05-04 01:32

本文选题：miniSTR + 复合扩增　；参考：《河北医科大学》2009年硕士论文

【摘要】： 目的:目前,高度降解检材的DNA分型一直是法医DNA领域的一大难题,面对命案现场或重大群体性灾难事件中的一些高度腐败降解检材,应用现有的短串联重复序列(short tandom repeats, STR)分型技术往往不能获得成功分型及明确的分型结果,为案件侦破或失踪人员的认定带来困难。针对这一问题,法医学家主要从样本DNA提取及分型技术两方面进行研究,如:研发或改进DNA提取技术,尽量获取大片段的DNA;应用SNP、miniSTR技术对小片段的DNA进行分型等。2001年9.11恐怖事件中miniSTR技术有力地推动了遇难者个人识别工作的进行,2003年Butler等首次提出miniSTR的概念,并正式命名为miniSTR技术。miniSTR技术就是在设计引物时,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样本检测可提高个人识别和亲权鉴定的检测成功率。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。目前,美国的AB公司已开发出商品化的minifiler试剂盒,其中包括D13S317、D7S820、D2S1338、D21S11、D16S539、D18S51、CSF1PO和FGA,以及性别基因Amelogenin,共九个基因座。minifiler试剂盒能够解决部分高度降解检材的DNA分型。为了增加系统效能,美国国家标准及技术研究院(National Institute of Standards andTechnology, NIST)研发了26个CODIS外的miniSTR基因座,其中,D10S1248、D14S1434、D22S1045三个基因座已被欧洲DNA数据库收录。美国、日本、西班牙、新加坡、韩国等国家也相继报道了miniSTR D1S1677、D4S2364、D10S1248等基因座的群体遗传学数据及其法医学应用价值,而我国相关研究较少且缺乏群体遗传学数据。为调查这些基因座在中国人群中的应用价值,开发国产miniSTR试剂盒,本研究对D10S1248、D2S441、D1S1677和D9S1122、D10S1435、D17S1301六个miniSTR基因座进行中国山东汉族人群群体遗传学调查,并构建两组荧光标记复合扩增体系,探讨其法医学应用价值,以期为将miniSTR技术应用于法医实践提供参考。方法:采用二次Chelex-100法提取120份山东汉族无关健康个体全血基因组DNA。本实验构建了2个复合扩增体系。复合扩增体系1包括D10S1248, D2S441, D1S1677三个miniSTR基因座,其上游引物5’端分别用6-FAM、HEX、TAMRA荧光标记;复合扩增体系2包括D9S1122、D10S1435、D17S1301三个miniSTR基因座,其上游引物5’端分别用6-FAM、HEX、TAMRA荧光标记。复合扩增反应体系为10μl,对反应体系中的引物浓度、Mg2+、Taq聚合酶、退火温度、循环次数等条件进行优化,得到最佳反应条件。PCR复合扩增产物在ABI PRISM 3130、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。依据本实验室构建的等位基因标准物及测序结果对各产物进行等位基因命名。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已确定亲缘关系的父-子-母三联体样本10例,对6个miniSTR基因座进行遗传稳定性的研究。将9947 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PowerPlex16试剂盒进行DNA分型,比较二者分型的成功率;将尸体解剖的皮肤、肌肉组织、肾脏、脾脏放置于37℃,100%湿度的孵箱中模拟降解检材,在一天、两天、四天、一周提取DNA,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PowerPlex16试剂盒进行DNA分型,比较二者分型的成功率。结果: 1、复合扩增体系的建立: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,6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。6个基因座的扩增片段长度均小于150bp。 2、群体遗传学调查结果: 120名山东汉族无关个体中D10S1248、D2S441、D1S1677、D9S1122、D10S1435、D17S1301基因座分别检出8、7、6、6、8、8个等位基因和20、18、12、17、20、22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0.750,0.775,0.650,0.708,0.750,0.858;多态性信息含量(PIC)分别依次为0.75,0.71,0.60,0.69,0.73,0.77;非父排除率(PE)分别依次为0.510,0.553,0.355,0.441,0.51,0.711;个人识别力(PD)分别依次为0.900,0.881,0.809,0.881,0.893,0.913。六个miniSTR基因座的累积非父排除概率为0.988817,累积个人识别力为0.9999975。 3、遗传稳定性分析结果:对10个已经确定亲权关系的三联体样本研究表明,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。 4、系统灵敏度研究结果:在DNA含量为0.1ng时,PowerPlex16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。 5、降解微量检材分析:对降解两个月的血液检材和在孵箱中模拟降解检材的人体组织,虽然结果中出现了少量杂峰,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。 6、种属特异性研究:一些常见动物如:猴、猪、牛、狗、兔、鱼未检测到特异性扩增产物。结论:D10S1248、D2S441、D1S1677、D9S1122、D10S1435和D17S1301六个miniSTR基因座在山东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。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,为开发国产化miniSTR分型试剂盒奠定了基础。
[Abstract]:Objective: at present, the DNA classification of highly degraded materials has been a major problem in the field of forensic DNA. In the face of some highly corrupted degradation materials in the scene of life or major mass disaster events, the application of the existing short tandem repeat (short tandom repeats, STR) typing technique often fails to achieve successful typing and clear typing results. For this problem, forensic scientists mainly study from two aspects of sample DNA extraction and typing technology, such as: R & D or improvement of DNA extraction technology, as far as possible to obtain large fragments of DNA; SNP, miniSTR technology for small fragments of DNA in the.2001 years of terror miniSTR The technology has greatly promoted the personal identification work of the victims. In 2003, Butler and so on first proposed the concept of miniSTR and formally named miniSTR technology.MiniSTR technology to shorten the length of the amplified fragment as close as possible to the core repeat sequence when the primers were designed, so that the highly degraded DNA sample test could improve personal knowledge. The European DNA profiling group, EDNAP) is defined as a new generation of genetic markers following STR (the European DNA profiling group, EDNAP). Currently, AB companies in the United States have developed commercialized Minifiler kits. And FGA, as well as the sex gene Amelogenin, a total of nine loci.Minifiler kits can solve the DNA classification of partially degraded samples. In order to increase the system efficiency, the National Institute of standards and Technology (National Institute of Standards andTechnology, NIST) developed 26 CODIS miniSTR genes. 434, three D22S1045 loci have been included in the European DNA database. The United States, Japan, Spain, Singapore, Korea and other countries have also reported the genetic data of miniSTR D1S1677, D4S2364, D10S1248 and other genetic bases and their forensic application value, while our research is less and lack of population genetic data.
In order to investigate the application value of these loci in Chinese population, the domestic miniSTR kits were developed. In this study, the six miniSTR loci of D10S1248, D2S441, D1S1677 and D9S1122, D10S1435, D17S1301 were investigated in the population genetics of Shandong Han population in China, and two groups of fluorescent labeling complex amplification systems were constructed to explore the application price of forensic medicine. Value in order to provide reference for applying miniSTR technology to forensic practice.
Methods: two Chelex-100 methods were used to extract the whole blood genome DNA. from 120 unrelated healthy individuals of Shandong Han. The composite amplification system 1 included three miniSTR loci of D10S1248, D2S441 and D1S1677, and the 5 'end of the upstream primers were labeled with 6-FAM, HEX, TAMRA, and 2 including D9S1122, respectively. D10S1435, D17S1301 three miniSTR loci, its upstream primers 5 'end were labeled with 6-FAM, HEX, TAMRA fluorescence respectively. The compound amplification reaction system was 10 mu L, and the primer concentration, Mg2+, Taq polymerase, annealing temperature and cycle times in the reaction system were optimized, and the optimal reaction conditions were obtained in ABI PRISM 3130. The PRISM 310 gene analyzer automatically completed the data collection of electrophoresis and electrophoresis results. The results were calculated using Genemapper 3.2 to calculate the relative size of the amplified product fragments and carry out the genotyping. The allele frequencies of each product were named according to the allelic standard and sequencing results constructed in our laboratory. The genetic stability of the 6 miniSTR loci was studied by the previous paternity test, which had been identified by this forensic identification center, which had identified the parent and parent three body samples of the relatives, and the 9947 DNA samples were diluted into 1ng, 0.5ng, 0.1ng, 0.05ng for sensitivity detection. In addition, the fresh blood was placed in the natural environment in summer. During the week, two weeks, four weeks, and eight weeks of simulated degradation, 6 miniSTR loci fluorescent tagged composite amplification systems and commercial PowerPlex16 kits were used for DNA typing, and the success rate of the two types was compared; the autopsy skin, muscle tissue, kidney, and spleen were placed at 37, 100% humidity incubator. DNA was extracted at one day, two days, four days and one week by simulated degradation test. The 6 miniSTR loci fluorescent tagged composite amplification system and commercialized PowerPlex16 kit were used for DNA typing, and the success rate of the two genotyping was compared.
Result:
1, the establishment of the composite amplification system: This study established two groups of fluorescent multiplex amplification to detect 6 miniSTR genotypes. The typing results showed that 6 miniSTR loci alleles obtained clear genotyping results, non specific type of non specific amplification products, and 6 gene loci. The exact length of the.6 locus can be achieved. The length of the amplified fragments is less than 150bp..
2, the results of population genetics survey: 120 unrelated individuals of Shandong Han, D10S1248, D2S441, D1S1677, D9S1122, D10S1435, and D17S1301 loci detected 8,7,6,6,8,8 alleles and 20,18,12,17,20,22 genotypes respectively. The genotype frequency distribution was conformed to Hardy-Weinberg balance (P0.05) by chi 2 test. Six loci were in Chinese Han population. The observation value of heterozygosity (Ho) was 0.750,0.775,0.650,0.708,0.750,0.858, and the polymorphism information content (PIC) was respectively 0.75,0.71,0.60,0.69,0.73,0.77, and the non parent exclusion rate (PE) was 0.510,0.553,0.355,0.441,0.51,0.711 respectively, and the individual recognition power (PD) division was 0.900,0.881,0.809,0.881,0.893,0.913. six miniSTR genes in turn. The cumulative non parent exclusion probability is 0.988817, and the cumulative personal recognition power is 0.9999975..
3, the results of genetic stability analysis: the study of three associated samples of 10 identified parental relationships showed that the parents' allele could be inherited steadily to the children, consistent with the Mendel's law of genetics.
4, the results of the system sensitivity study: when the content of DNA is 0.1ng, the output of the PowerPlex16 kit is low, and it is not easy to carry out the correct typing, but the miniSTR compound amplification system can also be used to classify the DNA of 0.05ng.
5, the analysis of degrading trace material: the blood samples degraded for two months and the human tissues that simulated degradation in the incubator, although a small number of hybrid peaks were found in the results, there was no interference to the results of the classification, and the complete classification was obtained in the six miniSTR loci, showing that the miniSTR technology was more than the conventional STR kit. Power.
6, species specific research: some common animals such as monkeys, pigs, cattle, dogs, rabbits and fish did not detect specific amplification products.
Conclusion: the six miniSTR loci of D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301 have good genetic polymorphism in the Han population in Shandong area, which conforms to the Mendel genetic rule. The results of the two groups of fluorescent labeling complex amplification systems established in this study are accurate, stable and reliable, with a sensitivity of 0.05ng, for the analysis of trace and degradation. The success rate of DNA 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. It can also be used as a useful supplement to the conventional STR kit, improve the system efficiency, and lay the foundation for the development of domestic miniSTR typing kits.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

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