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
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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。
Background and objective
For non-recombinant region of Human Y chromosome are paternally inherited with haplotype from father to son, Y chromosome STRs(Y short tandem repeat, Y-STRs) are the major tool of forensic genetics, and has the unique advantages in discrimination, paternity test, analysis of mixed samples, determination of identity of unknown male's remain, and analysis of mixed stain or organization mixture from different male, and also are the vital genetic markers to analyze population migration history and reconstruct of the pedigree with the same patrilineal ancestor. However, because of genetic specificity, many Y-STR loci should be used to improve the identification power of Y-STR. Contrasted with autosomal STR, Y-STR loci show characteristic haplotype in different populations and ethnic groups. Now the imported commercial kits of Y-STR were used to analyze biological evidences in our country. Therefore, it is necessary to select more stable, suitable and higher polymorphism Y-STR loci for Chinese populations, and to establish the Y-STR multiplex system with good identification power.
The new Y-STRs were studied to aaplied to improving the identification power of Y chromosome for forensic applications. 10 new Y-STR loci were selected; the primers of suitable Loci for MiniSTR were redesigned. The allelic sequences and hapolatypes of these Y-STRs were analyzed for providing the nomenclature of allele and the data of forensic application by investigating genetic polymorphism of 10 new Y-STR in Chaoshan Han population. The Y-STR multiplex system of silver stain containing suitable loci that were selected from these loci were established, moreover, we will study the feasibility according to the guidance of The Working Group on DNA Analysis Methods. We intend to establish the system with reliable results, higher capacity of discrimination.
Methods and material
10 new Y-STR loci (DYS522, DYS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594 and DYS598) were selected from GDB and the species identification of these loci was carried out by BLAT. The primers of DYS565, DYS568, DYS593, DYS594 and DYS598 loci were redesigned by Primers 3.0 and BLAT. DNA of 159 unrelated male individuals from Chaoshan Han population was amplified, and all loci were amplified by single-plex PCR. The PCR products of 10 Y-STR loci were genotyped using a non-denaturing polyacrylamide gel electrophoresis with a continuous buffer system, and the gels were sliver stained. The alleles of each Y-STR loci were sequenced, and nomenclatured according to the recommendation of International Society of Forensic Genetics. The allele and haplotype frequencies of 10 Y-STR loci were calculated by the counting method. The test of forensic applications of 10 Y-STR loci was performed using pedigree, female, domestic animal, and hair shaft samples. Suitable Y-STR loci were selected, and the multiplex system was established. The products of multiplex PCR were detected using a nondenaturing polyacrylamide gel electrophoresis with a continuous buffer system, and the gels were silver stain. The Y-STR multiplex systems were tested using different DNA content, different tissue samples and bloodstain on common matrixes.
Results
1. Y-STR specific analysis
Their sequences of 10 Y-STR loci can be found in human genome, and the same sequences weren’t found in animals′genomes by e-PCR. Female and animal genomic DNA was amplified, and the PCR procducts weren’t observed.
2. Electrophoresis typing
The PCR products of 10 Y-STR loci could be well separated with nondenaturing PAGE, and the single band of all loci was observed in all male samples.
3. Sequencing results
Except for DYS588 and DYS593, other Y-STR loci were STR with the simple repeats. The size of amplified fragment of 10 Y-STR loci ranged from 108bp to 372bp. The allelic DNA of DYS593 was shorter than that of other loci, and the allelic DNA of DYS522 was longer than that of other loci.
4. Population genetic data
A total of 7, 5, 5, 4, 6, 9, 8, 4, 4, 4 alleles was detected at DYS522, DYS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594, DYS598 loci in our male samples, respectively. The value of gene diversity (GD) ranged from 0.1434 at DYS565 to 0.7994 at DYS570. A total of 138 haplotypes were found, and the haplotype diversity was 0.9973. Standards error was 0.00069. None mutation was observed in the 30 father/son pairs.
5. Forensic application
The amplified products at DYS565, DYS568, DYS593 and DYS598 were less than 130bp. The hair shafts could be genotyped using the aforementioned 4 loci.
6. Multiplex PCR
Two Y-STR multiplex systems containing 5 Y-STR loci were established, and MultiplexⅠcontains three Y-STRs including DYS549, DYS556 and DYS594, and MultiplexⅡcontains two Y-STRs including DYS570 and DYS593. The results of electrophoresis showed that the PCR products of loci in two Y-STR multiplex systems were agree with single loci PCR products. The amplified products of two Y-STR multiplex systems could be genotyped using nondenaturing PAGE, and their genotypes were same as that of single-plex PCR. The minimum amount of DNA that could be detected was 0.5 ng. The genotype of different tissues of the body was same. The bloodstain, which attached to common matrix, could be genotyped, and the genotyping results of same sample were same. The statistical data of population genetics showed that 97 haplotypes were found, and the haplotype diversity is 0.99 (standards error is 0.0013) in 159 male individuals from Chaoshan Han population.
Conclusions
1. These Y-STR loci are single copy and Y chromosome specific loci, and can be used for mixed stain analysis and paternity test.
2. Alleles of 10 new Y-STR loci were sequenced, and the genetic polymorphisms of these loci were surveyed in Chaoshan Han population. The data provided the basis for comparing genetic data among different groups, and enriched the database of population genetics of Y-STR.
3. Haplotype diversity of 10 new Y-STR loci is 0.9973. Haplotype of 10 Y-STRloci has a better power of discrimination and excluding probability of paternity, and can improve the power of identification of Y chromosome.
4. Less than 130bp MiniSTR can improve genotype of STR of hair shaft, and can detect the seriously degraded DNA samples.
5. We successfully established two Y-STR multiplex systems with sliver stain, MultiplexⅠand MultiplexⅡ. Two Y-STR multiplex systems with reliable results, low cost and simple method have a good male specificity, species specificity and reproducibility, and high susceptivity. Bloodstain adhered to common material could be well genotyped with two Y-STR multiplex systems. Power of discrimination and excluding probability of paternity are 0.99 in two Y-STR multiplex systems, which provided a set Y-STR for improving the power of identification of Y chromosome. Two Y-STR multiplex systems are suitable for forensic applications.
6. The method of two Y-STR multiplex systems can provide reference for the establishment of other Y-STR multiplex systems.
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。
Background and objective
For non-recombinant region of Human Y chromosome are paternally inherited with haplotype from father to son, Y chromosome STRs(Y short tandem repeat, Y-STRs) are the major tool of forensic genetics, and has the unique advantages in discrimination, paternity test, analysis of mixed samples, determination of identity of unknown male's remain, and analysis of mixed stain or organization mixture from different male, and also are the vital genetic markers to analyze population migration history and reconstruct of the pedigree with the same patrilineal ancestor. However, because of genetic specificity, many Y-STR loci should be used to improve the identification power of Y-STR. Contrasted with autosomal STR, Y-STR loci show characteristic haplotype in different populations and ethnic groups. Now the imported commercial kits of Y-STR were used to analyze biological evidences in our country. Therefore, it is necessary to select more stable, suitable and higher polymorphism Y-STR loci for Chinese populations, and to establish the Y-STR multiplex system with good identification power.
The new Y-STRs were studied to aaplied to improving the identification power of Y chromosome for forensic applications. 10 new Y-STR loci were selected; the primers of suitable Loci for MiniSTR were redesigned. The allelic sequences and hapolatypes of these Y-STRs were analyzed for providing the nomenclature of allele and the data of forensic application by investigating genetic polymorphism of 10 new Y-STR in Chaoshan Han population. The Y-STR multiplex system of silver stain containing suitable loci that were selected from these loci were established, moreover, we will study the feasibility according to the guidance of The Working Group on DNA Analysis Methods. We intend to establish the system with reliable results, higher capacity of discrimination.
Methods and material
10 new Y-STR loci (DYS522, DYS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594 and DYS598) were selected from GDB and the species identification of these loci was carried out by BLAT. The primers of DYS565, DYS568, DYS593, DYS594 and DYS598 loci were redesigned by Primers 3.0 and BLAT. DNA of 159 unrelated male individuals from Chaoshan Han population was amplified, and all loci were amplified by single-plex PCR. The PCR products of 10 Y-STR loci were genotyped using a non-denaturing polyacrylamide gel electrophoresis with a continuous buffer system, and the gels were sliver stained. The alleles of each Y-STR loci were sequenced, and nomenclatured according to the recommendation of International Society of Forensic Genetics. The allele and haplotype frequencies of 10 Y-STR loci were calculated by the counting method. The test of forensic applications of 10 Y-STR loci was performed using pedigree, female, domestic animal, and hair shaft samples. Suitable Y-STR loci were selected, and the multiplex system was established. The products of multiplex PCR were detected using a nondenaturing polyacrylamide gel electrophoresis with a continuous buffer system, and the gels were silver stain. The Y-STR multiplex systems were tested using different DNA content, different tissue samples and bloodstain on common matrixes.
Results
1. Y-STR specific analysis
Their sequences of 10 Y-STR loci can be found in human genome, and the same sequences weren’t found in animals′genomes by e-PCR. Female and animal genomic DNA was amplified, and the PCR procducts weren’t observed.
2. Electrophoresis typing
The PCR products of 10 Y-STR loci could be well separated with nondenaturing PAGE, and the single band of all loci was observed in all male samples.
3. Sequencing results
Except for DYS588 and DYS593, other Y-STR loci were STR with the simple repeats. The size of amplified fragment of 10 Y-STR loci ranged from 108bp to 372bp. The allelic DNA of DYS593 was shorter than that of other loci, and the allelic DNA of DYS522 was longer than that of other loci.
4. Population genetic data
A total of 7, 5, 5, 4, 6, 9, 8, 4, 4, 4 alleles was detected at DYS522, DYS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594, DYS598 loci in our male samples, respectively. The value of gene diversity (GD) ranged from 0.1434 at DYS565 to 0.7994 at DYS570. A total of 138 haplotypes were found, and the haplotype diversity was 0.9973. Standards error was 0.00069. None mutation was observed in the 30 father/son pairs.
5. Forensic application
The amplified products at DYS565, DYS568, DYS593 and DYS598 were less than 130bp. The hair shafts could be genotyped using the aforementioned 4 loci.
6. Multiplex PCR
Two Y-STR multiplex systems containing 5 Y-STR loci were established, and MultiplexⅠcontains three Y-STRs including DYS549, DYS556 and DYS594, and MultiplexⅡcontains two Y-STRs including DYS570 and DYS593. The results of electrophoresis showed that the PCR products of loci in two Y-STR multiplex systems were agree with single loci PCR products. The amplified products of two Y-STR multiplex systems could be genotyped using nondenaturing PAGE, and their genotypes were same as that of single-plex PCR. The minimum amount of DNA that could be detected was 0.5 ng. The genotype of different tissues of the body was same. The bloodstain, which attached to common matrix, could be genotyped, and the genotyping results of same sample were same. The statistical data of population genetics showed that 97 haplotypes were found, and the haplotype diversity is 0.99 (standards error is 0.0013) in 159 male individuals from Chaoshan Han population.
Conclusions
1. These Y-STR loci are single copy and Y chromosome specific loci, and can be used for mixed stain analysis and paternity test.
2. Alleles of 10 new Y-STR loci were sequenced, and the genetic polymorphisms of these loci were surveyed in Chaoshan Han population. The data provided the basis for comparing genetic data among different groups, and enriched the database of population genetics of Y-STR.
3. Haplotype diversity of 10 new Y-STR loci is 0.9973. Haplotype of 10 Y-STRloci has a better power of discrimination and excluding probability of paternity, and can improve the power of identification of Y chromosome.
4. Less than 130bp MiniSTR can improve genotype of STR of hair shaft, and can detect the seriously degraded DNA samples.
5. We successfully established two Y-STR multiplex systems with sliver stain, MultiplexⅠand MultiplexⅡ. Two Y-STR multiplex systems with reliable results, low cost and simple method have a good male specificity, species specificity and reproducibility, and high susceptivity. Bloodstain adhered to common material could be well genotyped with two Y-STR multiplex systems. Power of discrimination and excluding probability of paternity are 0.99 in two Y-STR multiplex systems, which provided a set Y-STR for improving the power of identification of Y chromosome. Two Y-STR multiplex systems are suitable for forensic applications.
6. The method of two Y-STR multiplex systems can provide reference for the establishment of other Y-STR multiplex systems.
引文
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[18] Hellmann A, Rohleder U, Schmitter H, et al. STR typing of human telogen hairs a new approach[J]. Int J Legal Med,2001,114(4-5):269-273.
[19] Butler JM, Shen Y, McCord BR. The development of reduced size STR amplicons as tools for analysis of degraded DNA[J]. Forensic Sci,2003,48(5):1054-1064.
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[23] Walsh BS, Petzger DA, Higuchi R. Chelex-100 as medium for simple extraction of DNA for PCR-based typing from forensic material[J]. Biotechniques,1991,10(4):506-513.
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[31] Hanson EK, Ballantyne J. Comprehensive annotated STR physical map of the human Y chromosome: Frensic implications[J]. Leg Med,2006,8(2):110-120.
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