四个X-STR基因座四色荧光复合分型体系构建及群体遗传学调查

发布时间：2018-04-23 12:52

本文选题：X染色体 + 短串联重复序列　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:人类X染色体STRs是指存在于X染色体上特异碱基短串联重复片段。近年来X染色体STRs因其高稳定性,高多态性,独特的遗传方式,在解决特殊案件中无法比拟的优点而逐渐受到重视,虽然早在1991年就有HPRTB,ARA和DXS981等X-STR基因座的报道,但当前已获得群体遗传学数据且可用于法医遗传学分析的X-STR基因座仅有30多个,X染色体STRs群体遗传学数据严重欠缺,数量不足,存在人群和种族差异,基因座间连锁关系又限制其使用,因此研究寻找更多能为法医学所使用的X-STR基因座,显得尤为重要。本课题旨在选取4个未报道群体遗传特征的X-STR基因座,即GATA151A05/GATA2B05/DXS7129/ HUMUT1595,并构建荧光标记复合扩增体系,对北方汉族人群进行群体遗传学调查,为开发国产X-STRs试剂盒、DNA数据库建立和群体遗传学研究提供基础资料。方法: 1基因座的选择:选择未报道群体遗传学特征且位于X染色体上物理距离较近(10Mb),位于同一连锁群内的4个X-STR基因座。 2复合分型体系的构建:对GATA2B05、HUMUT1595、GATA151A05、DXS7129这4个已选择的X-STRs进行PCR复合扩增。在GATA2B05、HUMUT1595基因座的上游引物5’端标记6-FAM荧光,GATA151A05基因座的上游引物5’端标记HEX荧光,DXS7129基因座的上游引物5’端标记ROX荧光。优化各基因座引物浓度比例、Mg2+和Taq酶浓度、退火温度、循环次数等因素。应用ABI310及ABI3130型遗传分析仪对扩增产物进行检测,Genemapper 3.2软件分析结果并对样本进行检测分型。各基因座每一种等位基因片段随机选择一个男性样本进行测序,依据测序结果并根据国际法医遗传学会(international society of forensic genetics, ISFG)推荐的命名原则对各等位基因进行命名。 3群体遗传学调查和法医学应用:应用已复合的扩增体系对北方汉族214例健康无关个体(男105例,女109例)进行群体遗传学调查。结果用Arlequin3.11软件进行分析,使用http://www.chrx-str.org网站在线计算相关法医遗传学参数。并对本室日常检案中的10例单亲鉴定案件(4例父女单亲鉴定案件,6例母子单亲鉴定案件)进行了调查,及本法医病理教研室尸检标本进行了组织同一性检验。结果: 1荧光复合分型体系构建:成功建立了GATA151A05 /GATA2B05 /DXS7129 / HUMUT1595四个X-STR基因座荧光标记复合扩增体系。20μl反应体系中含引物GATA151A05、GATA2B05、DXS7129、HUMUT1595分别为6pmol、5pmol、4pmol、5pmol,Taq DNA聚合酶1U,MgCl2浓度1.5mM,采用热启动的方式进行扩增。该体系各基因座间峰型均匀、杂合子扩增均衡、无非特异性扩增、重复性好。 2群体遗传学调查结果:应用该体系对北方汉族214例无关个体进行了群体遗传学调查。女性109例样本中,基因座DXS7129检出5个等位基因和8种基因型;GATA151A05检出5个等位基因和9种基因型;HUMUT1595检出8个等位基因和14种基因型;GATA2B05检出8个等位基因和18种基因型;男性105例样本中,基因座DXS7129检出5个等位基因;GATA151A05检出6个等位基因;HUMUT1595检出7个等位基因;GATA2B05检出8个等位基因。对各基因座进行哈温平衡检验,除GATA2B05基因座不符合哈温平衡外,其余三个基因座均符合哈温平衡。对符合哈温平衡的DXS7129,GATA151A05,HUMUT1595基因座进行了测序,男女等位基因频率差异性检测,基因座间连锁不平衡分析和遗传参数计算。这3个基因座各等位基因在男女之间无差别,合并计算等位基因频率(0.00309—0.7678),基因座连锁不平衡分析显示3个基因座之间相互独立。3个X-STR基因座DXS7129、HUMUT1595、GATA151A05在北方汉族人群中观察值杂合度(Ho)分别依次为:0.29358、0.60550、0.54128,多态信息量(PIC)分别依次为:0.36858、0.62258、0.58478,女性个体排除率(PDfemale)分别依次为:0.593145、0.761857、0.731926,男性个体排除率(PDmale)分别依次为:0.385692、0.602436、0.573134 ,三联体非父平均排除概率(MEC Kishida)分别依次为: 0.356211、0.522350、0.487274,二联体非父平均排除概率(MEC Desmarais Duo)分别依次为: 0.226958、0.380390、0.346576。 3家系调查:在对本室日常检案中的10例单亲鉴定案件(4例父女单亲鉴定案件,6例母子单亲鉴定案件)的检测中未发现基因座突变,且这四个X-STR基因座是按照X染色体标记物特有的交叉遗传方式遗传。 4组织同一性检测:该体系对同一个体的心脏、肝脏、肾脏、肌肉组织检测结果分型一致。结论:本研究建立了一组法医学遗传标记GATA151A05 /GATA2B05 /DXS7129 HUMUT1595四色荧光标记复合扩增体系,该体系分型准确、重复性好。对该体系的群体遗传学调查表明: HUMUT1595具有高度多态性,GATA151A05具有中度多态性,可用于法医学亲子鉴定和个人识别,充实了尚欠缺的X-STRs群体遗传学数据,可为开发国产X-STRs试剂盒、建立X-STRs数据库建立提供基础资料。
[Abstract]:Objective: human X chromosome STRs refers to the specific base short tandem repeats existing on the X chromosome. In recent years, the X chromosome STRs has been gradually valued for its high stability, high polymorphism and unique genetic method, which is unparalleled in solving special cases, although the X-STR loci such as HPRTB, ARA and DXS981 have been reported early in 1991. But there are only more than 30 X-STR loci that have obtained population genetic data and can be used for forensic genetic analysis. The X chromosome STRs population genetic data is deficient, the number is insufficient, the population and race differences exist, and the interloci linkage relationship restricts its use. This study seeks to find more X-STR bases used for forensic medicine. Because of the seat, it is particularly important.
The purpose of this project is to select 4 X-STR loci, that is, GATA151A05/GATA2B05/DXS7129/ HUMUT1595, which is the genetic feature of the unreported population, and to construct a fluorescence labelled composite amplification system to investigate the population genetics of the Han population in the north, and provide the basic information for the development of domestic X-STRs kits, the establishment of DNA database and the study of population genetics.
Method:
1 loci selection: select the unreported population genetic characteristics and the physical distance on the X chromosome is close (10Mb), located in the same linkage group of 4 X-STR loci.
2 the construction of the 2 compound typing system: PCR composite amplification of 4 selected X-STRs for GATA2B05, HUMUT1595, GATA151A05, DXS7129. In GATA2B05, the upstream primers of the HUMUT1595 loci are labeled 6-FAM fluorescence, the upstream primer 5 'of the GATA151A05 loci are labeled HEX fluorescence, and the upstream primers of the DXS7129 genre are labeled with the 5' end of the DXS7129 genre. The ratio of primer concentration, Mg2+ and Taq enzyme concentration, annealing temperature, and cycle times were optimized. ABI310 and ABI3130 genetic analyzer were used to detect the amplification products, Genemapper 3.2 software analysis results and samples were detected. Each allele fragment of each loci was randomly selected for a male sample. Sequenced, the alleles were named according to the sequencing results and based on the naming principles recommended by the international society of forensic genetics (ISFG).
The 3 population genetics survey and forensic application: the population genetics survey of 214 unrelated health individuals (105 men and 109 women) of the Han nationality in the north of the northern Han Dynasty was investigated with a compound amplification system. The results were analyzed by Arlequin3.11 software, and the relevant forensic genetic parameters were calculated online by the http://www.chrx-str.org website. 10 cases of single parent identification (4 cases of paternity single parent identification, 6 parent parent parent identification cases) were investigated, and the autopsy specimens of this forensic pathology teaching and research department were tested for the same identity.
Result:
The construction of 1 fluorescent composite typing system: the GATA151A05 /GATA2B05 /DXS7129 / HUMUT1595 /DXS7129 / HUMUT1595 fluorescent labeling complex amplification system was established, and the primers GATA151A05, GATA2B05, DXS7129, HUMUT1595, respectively, were used as the thermal startup method. The amplify of the loci was homogeneous, heterozygote amplification was balanced, and the amplification was not only non specific amplification, but also good reproducibility.
The results of the 2 population genetics survey: using this system to investigate the population genetics of 214 unrelated individuals in the northern Han population, 5 alleles and 8 genotypes were detected in 109 female samples, 5 alleles and 9 genotypes were detected by GATA151A05, and 8 alleles and 14 genotypes were detected by HUMUT1595; and 8 was detected by GATA2B05. 8 18 alleles and 5 alleles were detected in 105 male samples, 5 alleles were detected by DXS7129, 6 alleles were detected by GATA151A05, 7 alleles were detected by HUMUT1595, and 8 alleles were detected by GATA2B05. The rest of the gene pedestal, except that the GATA2B05 locus was incompatible with the hash balance, had the other three loci. The DXS7129, GATA151A05, HUMUT1595 loci were sequenced, the allele frequency differences of male and female alleles, interloci linkage disequilibrium analysis and genetic parameters calculation. The alleles of the 3 loci were not different between men and women, and the frequencies of alleles (0.00309 - 0.7678) were combined (0.00309 - 0.7678). The linkage disequilibrium analysis showed that the independent.3 X-STR locus DXS7129, HUMUT1595, and GATA151A05 in the northern Han population were respectively 0.29358,0.60550,0.54128, and the polymorphic information (PIC), respectively, was 0.36858,0.62258,0.58478, and the female individual exclusion rate (PDfemale) was respectively dependent on the 3 loci. The following is: 0.593145,0.761857,0.731926, the male individual exclusion rate (PDmale), respectively, is 0.385692,0.602436,0.573134, and the non parent average exclusion probability (MEC Kishida) of the three body (MEC Kishida) is respectively 0.356211,0.522350,0.487274, and the non parent average exclusion probability (MEC Desmarais Duo) of the two body (MEC Desmarais Duo) is respectively 0.226958,0.380390,0.346576..
3 survey: 10 single parent identification cases (4 cases of paternity parental identification, 6 parent parent identification cases) were not detected in the laboratory examination, and the four X-STR loci were inherited according to the characteristic cross inheritance of X chromosome markers.
4 tissue identity test: the system is identical to the heart, liver, kidney and muscle tissue of the same individual.
Conclusion: a group of forensic genetic markers GATA151A05 /GATA2B05 /DXS7129 HUMUT1595 four color fluorescent labeling complex amplification system has been established in this study. The system is accurate and reproducible. The population genetic investigation of this system shows that HUMUT1595 has high polymorphism and GATA151A05 has moderate polymorphism, which can be used in forensic parents. Identification and personal identification have enriched the X-STRs population genetic data which are not yet available. It can provide basic information for developing domestic X-STRs kit and establishing X-STRs database.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：D919

【参考文献】