潮汕地区汉族群体10个新Y染色体STR基因座的遗传多态性及复合扩增研究

发布时间：2018-06-06 09:38

本文选题：Y染色体 + 短串联重复序列　；参考：《汕头大学》2009年硕士论文

【摘要】： 背景与目的人类Y染色体的非重组区(non-recombination regions of Y chromosome,NRY)为单倍型父系遗传,这使得Y染色体STR (Y short tandem repeat,Y-STR)在法医学个人识别、父权鉴定、男女性混合检材检测、无名男尸的身源确定、不同男性个体混合斑或组织混合物的分析,以及群体迁徙、人类进化及父系家族分析中具有独特的优势,已成为目前法医学检验的主要遗传标记。但由于遗传上的特殊性,与常染色体STR相比,Y-STR单倍型分布具有明显的群体特异性,且Y-STR的个人识别能力和非父排除率也远远低于常染色体STR,要提高Y染色体的鉴别能力,必须尽可能多的联合使用Y-STR基因座。目前,国内主要依赖进口Y-STR试剂盒分析法医物证检材,其所包含的Y-STR基因座数量有限,且由于基于国外白种人开发,在一定程度上难以满足我国法医实际工作需要。因此有必要筛选出更多稳定、多态性好、适合我国人群的Y-STR基因座,并构建具有较高鉴定能力的Y-STR复合扩增体系。本研究旨在筛选更多新的Y-STR基因座,提高Y-STR单倍型的鉴别能力;选取10个新Y-STR基因座,对其中适合MiniSTR引物设计的基因座,进行MiniSTR引物设计;通过对10个Y-STR基因座在潮汕地区汉族群体中的遗传多态性调查,分析它们的等位基因序列和单倍型,为等位基因命名提供依据,为法医学应用提供基础数据;根据群体调查结果,选出其中符合条件的基因座,应用银染复合扩增技术,组建Y-STR复合扩增体系,依据DNA分析技术工作组(the working group on DNA analysis methods,TWGDAM)指南进行法医学可行性研究,以建立检测结果可靠、具有较高个人识别能力的检测方法。材料与方法利用BLAT (BLAST-like alignment tool)软件对GDB数据库(genome database)中的Y-STR基因座进行种属特异性分析,依据具有良好种属特异性、重复序列为单拷贝、重复单位为四或五核苷酸的原则,筛选出10个新Y-STR基因座DYS522、YS549、DYS556、DYS565、DYS568、DYS570、DYS588、DYS593、DYS594、DYS598;用primer 3.0软件和BLAT软件,根据Y-MiniSTR引物设计原则,对DYS565、DYS568、DYS593、DYS594和DYS598基因座重新进行MiniSTR引物设计,其它基因座引物则采用GDB的引物序列。应用PCR反应,对实验DNA样本进行单基因座PCR扩增,扩增产物采用非变性聚丙烯酰胺凝胶连续缓冲体系垂直电泳分型,银染法显色检测;用女性样本和常见动物样本,实验检测10个Y-STR基因座的Y染色体特异性和种属特异性;对潮汕地区汉族159名无关男性个体样本进行分型检测,各基因座的等位基因测序后,按国际法医遗传学会(ISFG)推荐的命名原则命名;采用直接计数法计算10个Y-STR基因座的等位基因频率和单倍型频率;用家系样本、毛干样本DNA作为模板,检测Y-STR基因座的突变率和分析降解DNA的能力。在群体调查结果基础上,依据银染复合扩增位点选择的原则,选出符合条件的Y-STR基因座,通过优化复合扩增条件,建立复合扩增体系;扩增产物应用非变性聚丙烯酰胺凝胶连续缓冲体系垂直电泳分型、银染显色检测;用不同DNA含量样本、不同组织样本、不同载体上的血痕作模板,对复合扩增体系进行测试。结果 1 Y-STR特异性分析结果应用e-PCR对10个基因座进行种属特异性分析,各基因座的DNA序列只与人类基因组DNA完全匹配,未见其他动物基因组DNA中存在相同序列;用10个Y-STR基因座的引物,扩增女性样本和常见动物样本DNA,均未检测到扩增产物。 2电泳分型 10个Y-STR基因座的扩增产物均只检测到一条带,非特异性产物少,分型效果良好。 3等位基因测序结果 10个Y-STR基因座除DYS588和DYS593外,其余均为简单重复序列Y-STR。它们在潮汕地区汉族人群中的扩增片段长度在108~372bp之间,其中DYS593扩增片段长度最小,DYS522最大。 4群体遗传学数据潮汕地区汉族159名无关男性个体中,DYS522、YS549、DYS556、DYS565、DYS568、DYS570、DYS588、DYS593、DYS594、DYS598基因座分别检测出7、5、5、4、6、9、8、4、4、4个等位基因,基因变异度在0.1434 (DYS565)~0.7994 (DYS570)之间;10个Y-STR基因座,共观察到138种不同的单倍型,单倍型变异度为0.9973,标准误为0.00069。通过30个2代父子家系样本分析,10个Y-STR基因座单倍型一致,未观察到突变。 5法医学应用结果 10个Y-STR基因座中扩增片段长度小于130bp的DYS565、DYS568、DYS593、DYS598基因座,可对毛干DNA进行检测分型。 6复合扩增结果选出了5个Y-STR基因座建立两组Y-STR复合扩增银染检测体系:MultiplexⅠ为DYS549、DYS556和DYS594,MultiplexⅡ为DYS570和DYS593;电泳结果显示:两组Y-STR银染复合扩增体系的各基因座间扩增产物平衡,非变性聚丙烯酰胺凝胶电泳后可对其进行准确分型,且与单基因座扩增结果一致。法医学应用研究结果显示:两组复合扩增体系具有良好的男性特异性和较高的种属特异性,最低DNA检出量达0.5ng;同一个体不同组织器官DNA分型结果一致;对涂在不同载体上的血痕可进行分型,同一样本分型一致。经群体遗传学数据统计,5个基因座在潮汕地区汉族159名男性样本中共观察到97种单倍型,单倍型变异度为0.99,标准误为0.0013。结论 1本研究筛选的10个Y-STR基因座都是单拷贝的Y染色体特异性STR基因座,具有很好的种属特异性和稳定的父系遗传,可用于法医学混合斑迹分析和父权关系的鉴定。 2分析了10个Y-STR基因座的等位基因序列,调查了它们在潮汕地区汉族人群中的遗传多态性,为比较不同群体间的数据提供了依据,丰富了我国人类遗传学数据库。 3 10个Y-STR基因座的单倍型变异度为0.9973,其构成的单倍型具有较高的个人识别率和非父排除率,能够有效提高Y染色体的鉴别能力。 4证实了小于130bp的小片段MiniSTR可对毛干DNA进行检测分型,提示对于高度降解的DNA检材能够准确分型。 5建立了两组Y-STR复合扩增银染检测体系:MultiplexⅠ和MultiplexⅡ;两组体系检测结果可靠,价格低廉;具有很好的男性特异性、种属特异性、系统重复性,灵敏度高;对附着在各种常见载体上的血痕具有良好的检测分析能力;个人识别率和非父排除率为0.99,为提高Y染色体的鉴别能力提供了一种经济、快速、高效的检测方法,可用于法医学实际检案。 6本实验Y-STR复合扩增体系建立的方法,可作为其他Y-STR基因座银染和荧光复合扩增的参考依据。
[Abstract]:Background and purpose
The non recombinant region of the human Y chromosome (non-recombination regions of Y chromosome, NRY) is a haplotype paternal inheritance. This makes Y chromosome STR (Y short tandem) in forensic personal identification, paternity testing, male and female mixed detection, the body source of unnamed male corpse, mixed spots or tissue mixtures of different male individuals. Analysis, population migration, human evolution and paternal family analysis have unique advantages, and have become the main genetic markers of forensic examination. But because of the genetic particularity, the distribution of Y-STR haplotypes has distinct population specificity compared with the autosomal STR, and the individual recognition and non parent exclusion of Y-STR are also far away. Below the autosomal STR, in order to improve the identification ability of the Y chromosome, it is necessary to use as many Y-STR loci as possible. At present, the domestic mainly rely on the import Y-STR kit to analyze forensic material, which contains a limited number of Y-STR loci, and it is difficult to meet the legal practice of our country to a certain extent because of the development of the foreign white people. It is necessary to work. Therefore, it is necessary to screen more stable, polymorphic, suitable Y-STR loci in the population of our country, and to construct a complex Y-STR amplification system with high identification ability.
The purpose of this study is to screen more new Y-STR loci and improve the identification ability of Y-STR haplotype. 10 new Y-STR loci are selected to design the MiniSTR primer which is suitable for MiniSTR primers, and the genetic polymorphism of 10 Y-STR loci in the Han population in Chaoshan region is analyzed, and their allele sequence is analyzed. Column and haplotype, provide basis for allele naming, provide basic data for forensic application, select the suitable loci according to the results of group survey, use silver dye compound amplification technology to form Y-STR compound amplification system, according to the guide of DNA analysis technology working group (the working group on DNA analysis methods, TWGDAM) guide The feasibility study of forensic medicine is to establish a reliable detection method with high personal identification ability.
Materials and methods
The specific analysis of Y-STR loci in GDB database (Genome Database) was carried out by BLAT (BLAST-like alignment tool) software. According to the principle of good species specificity, repeat sequence as single copy and repeat unit as four or five nucleotides, 10 new Y-STR loci were screened for DYS522, YS549, DYS556. YS588, DYS593, DYS594, DYS598; using primer 3 software and BLAT software to design primers for DYS565, DYS568, DYS593, DYS594 and DYS598 genes, and the primers of other loci primers. A non denatured polyacrylamide gel continuous buffer system was used for vertical electrophoresis, silver staining and color detection. The Y chromosome specificity and species specificity of 10 Y-STR loci were tested with female samples and common animal samples, and 159 unrelated male body samples in Chaoshan region were detected by typing, and the alleles of each loci were found. After sequencing, it was named according to the naming principle recommended by the international forensic genetic association (ISFG); the allele frequency and haplotype frequency of 10 Y-STR loci were calculated by direct counting. The mutation rate of Y-STR loci and the ability to analyze the degradation of DNA were detected by family samples and DNA as a template. Based on the results of group investigation, the results were based on the results of group investigation. According to the principle of composite amplification site selection of silver staining, the Y-STR gene pedestal was selected, and the composite amplification system was established by optimizing the complex amplification conditions. The amplified products used the non denaturing polyacrylamide gel continuous buffer system for vertical electrophoresis and silver staining detection, with different DNA content samples, different tissue samples and different carriers. Blood samples were used as templates to test the multiplex amplification system.
Result
1 Y-STR specificity analysis results
The specific analysis of 10 loci was carried out by e-PCR. The DNA sequence of each loci was only completely matched with the human genome DNA, and the same sequence was not found in the other animal genome DNA, and the primers of 10 Y-STR loci were used to amplify the female samples and common animal samples DNA. All the amplified products were not detected.
2 electrophoretic typing
Only one band was detected in the amplified products of the 10 Y-STR loci. The nonspecific products were few and the typing results were good.
3 allele sequencing results
The 10 Y-STR loci, except for DYS588 and DYS593, were all simple repeat sequence Y-STR.. The length of the amplified fragment in the Han population in Chaoshan region was between 108~372bp, of which the length of DYS593 amplification fragment was the smallest and the DYS522 was the largest.
4 population genetic data
Among 159 unrelated male individuals in the Chaoshan region, DYS522, YS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594, DYS598 loci respectively detected the 7,5,5,4,6,9,8,4,4,4 allele, and the gene variation was 0.1434 (DYS565), and 138 different haplotypes, haplotypes, were observed. The dissimilarity is 0.9973, the standard error is 0.00069.. Through the analysis of 30 2 generation father and son family samples, 10 Y-STR loci haplotypes are identical, no mutation is observed.
5 application results of Forensic Medicine
In the 10 Y-STR loci, DYS565, DYS568, DYS593 and DYS598 loci with a fragment length less than 130bp can be used for detection and typing of hairy stem DNA.
6 compound amplification result
5 Y-STR loci were selected to establish two groups of Y-STR composite amplification silver staining detection system: Multiplex I was DYS549, DYS556 and DYS594, Multiplex II was DYS570 and DYS593. The electrophoresis results showed that the amplification products of the two groups of Y-STR silver staining complex amplification system were balanced, and the non variable polyacrylamide gel electrophoresis could be used to accurately distinguish them. The results of the forensic application study showed that the two groups of composite amplification systems had good male specificity and higher species specificity, and the lowest DNA detection amount was 0.5ng; the DNA typing of different tissues and organs of the same individual was consistent; the blood stains on different carriers could be typed and the same sample was found. According to the data of population genetics, 97 haplotypes were observed by 5 loci in 159 male samples of Han nationality in Chaoshan region. The haplotype variation was 0.99, and the standard was 0.0013.
conclusion
1 the 10 Y-STR loci screened in this study are single copies of the Y chromosome specific STR loci, which have good genera specificity and stable paternal inheritance, which can be used for forensic mixed spot trace analysis and paternity identification.
2 the allelic sequence of 10 Y-STR loci was analyzed and their genetic polymorphism in the Han population in Chaozhou and Shantou area was investigated, which provided a basis for comparing the data between different populations, and enriched the database of human genetics in China.
The haplotype variation of the 310 Y-STR loci was 0.9973. The haplotypes made up of the haplotypes had a high individual recognition rate and a non parent exclusion rate, which could effectively improve the identification ability of the Y chromosome.
4 it was confirmed that the small fragment MiniSTR smaller than 130bp could be used for typing and typing of hairy stem DNA, suggesting that the highly degraded DNA can be accurately typed.
5 set up two groups of Y-STR composite amplification silver staining detection system: Multiplex I and Multiplex II; two groups of system detection results are reliable, low price, and have good male specificity, species specificity, system repeatability, high sensitivity; the blood marks attached to the various common carriers have good detection and analysis ability; individual recognition rate and non The parent exclusion rate is 0.99, which provides an economical, fast and efficient detection method to improve the identification ability of Y chromosome, and can be applied to forensic practice.
6 the method of establishing Y-STR multiplex amplification system can be used as reference basis for silver staining and fluorescence multiplex amplification of other Y-STR loci.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

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