法医线粒体DNA分型及中国不同民族多态性研究
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摘要
目的 为了提高线粒体DNA这一遗传标记的法医学鉴别能力，设计适合法医学检材的、覆盖mtDNA控制区的扩增引物，建立新的扩增与测序方法；应用所建立的方法调查多民族mtDNA控制区多态性，为涉及不同民族的mtDNA序列分析提供群体遗传学基础；应用获得的数据进行分子进化分析，探讨各民族间的亲缘关系及同一民族内的分子进化问题。方法 根据mtDNA控制区及其周围区域的序列，设计多对引物，探索优化扩增体系，使扩增条件能够同时满足多对引物的需要，用Sanger末端终止法及荧光标记技术对样本进行DNA测序，Sequencing Analysis3.4和Seq／Ede软件进行序列分析和比对。用毛发、指甲、微量血痕及各种陈旧骨骼样本对所建立的方法进行法医学有效性测试，并用实际案例验证其实用性；应用所建立方法对汉族、黎族、维吾尔族、瑶族、藏族无关个体样本共446份进行序列分析，调查不同民族mtDNA多态性；应用MEGA 2软件对所得各民族数据做进化距离分析，并构建各民族内部和民族间的系统发育树，探讨各民族间的遗传关系。结果 设计了覆盖整个mtDNA控制区及周围区域的5对引物，使各段扩增产物长度在299bp到452bp之间，统一了扩增条件使5段序列可以在相同循环参数下扩增。对陈旧骨骼、毛发、指甲等法医学样本均
中文摘要
得到可靠结果。该方法可以对细胞总DNA为0.01 sng的样本进行测序。
通过对不同民族血样的序列测定和统计学分析，分别在汉族、黎族、维
吾尔族、瑶族、藏族群体中各检出269、316、141、127、159个突变位
点;各民族平均变异数分别为15.3、15.9、12.0、10.5、11.3，在所用群
体样本中，总变异度分别为1、l、0.9967、0.9936、l。分析各民族高变
区I、高变区n和高变区nl的鉴别能力，鉴别能力均为高变区I>高变
区n>高变区Hl，而且各民族间多态性存在差异。本研究还发现不同
民族的高变区111所在区域不完全相同;同一民族不同地域、不同多态
J性区域的碱基变异也存在差异。在测定的无关个体mtDNA单倍型序列
的基础上做进化距离分析，并构建了各民族内部和民族间的系统发育
树。结论本课题所建立的线粒体DNA序列分析体系是一种对法医疑
难检材进行鉴别的有效、实用的方法，并且本方法已经在实际案件检验
中得到验证。对汉族、黎族、维族、瑶族、藏族群体进行序列分析结果
表明，将检测区域从HVI和HVH扩大到覆盖整个控制区及其周围区域
后大大提高了mtDNA这一遗传标记在法庭科学领域的鉴别能力。同时
在研究过程中检出的新的高变SNP和STR位点也为今后应用mtDNA进
行疑难样本的筛选提供了新的标记。各民族之间多态性比较发现民族间
存在差异，提示应根据不同民族样本建立参照数据库。5个民族之间的
遗传关系分析表明，所选择的群体样本及测序区能够满足群体遗传学分
析的要求，而且新发现的变异位点可以为建立更完善的系统树提供指
标。
Objective In order to improve the discrimination power of mitochondrial DNA in forensic DNA typing, primer pairs which covered the mtDNA control region were designed. The methods of both amplification and DNA sequencing with new primers were established. Polymorphisms of mtDNA control region in 5 ethnic groups in China were analyzed using the established methods to provide a basic database. Molecular evolution and phylogenetic issues were discussed based on the mtDNA database to understand genetic relationship of different ethnic groups in China. Methods The primers were design according to the Anderson's sequence. The amplification system was optimized so that the PCR with different primers can be carried out under the same condition. The PCR products were sequenced using
Sanger's terminator and fluorescent label techniques. Sequences were analyzed and compared base on Sequencing Analysis3.4 and Seq/Ede software. Validation study of forensic science was carried out using forensic samples such as hair, nail and badly degrade bone. The method has also been used in case work to validate the practicability. MAGA2 software was employed to analyze the genetic distance between samples and construct the phylogenentic tree. Result A total of five primer pairs was designed to cover the whole mtDNA control region and the neighbor part. The length of amplicon was from 299 bp to 452bp with different primer pairs. The successful result was obtained even if the DNA template was small to 0.015ng. The mtDNA typing of hair, nail and bone can be carried out with higher successful rate. Sequence analysis revealed that the number of variation site was 269, 316, 141, 117, 159 in Han, Li, Wei, Yao, Zang ethnic group, respectively, while the mean number of base pair with difference was 15.3, 15.9, 12.0, 10.5, 11.3 in Han, Li, Wei, Yao, Zang ethnic group, respectively. The genetic diversity of population samples from Han, Li, Wei, Yao, Zang ethnic group was 1,1, 0.9967, 0.9936, 1, respectively. The result of analysis for discrimination power from three hypervariable regions showed I higher than II and II higher than III. The third hypervariable region was different among the ethnic groups we studied. The analysis of variation in Han group revealed geographic difference. The genetic distances based on the sequence of mtDNA haplotype
have been obtained and the phylogenetic trees were constructed. Conclusion The mtDNA sequencing system using new primers was useful for forensic purpose. For difficult samples, DNA typing with this system was especially available and practical, and has been validated in criminal cases. After extending the sequenced region tb whole control region, the sequence in Han, Li, Wei, Yao, Zang ethnic groups revealed that the discrimination power of mtDNA was increased greatly. The novel STR and SNP loci found in the region of mtDNA also provide useful markers for screening test of forensic samples. The different polymorphisms among ethnic groups imply that it is necessary to establish reference database based on each ethnic group. The study of genetic relationship showed that the selected samples and the sequenced mtDNA region satisfied the request of the genetic analysis. The novel variable site provided some index to establish a perfect phylogenetic tree.
中文摘要
得到可靠结果。该方法可以对细胞总DNA为0.01 sng的样本进行测序。
通过对不同民族血样的序列测定和统计学分析，分别在汉族、黎族、维
吾尔族、瑶族、藏族群体中各检出269、316、141、127、159个突变位
点;各民族平均变异数分别为15.3、15.9、12.0、10.5、11.3，在所用群
体样本中，总变异度分别为1、l、0.9967、0.9936、l。分析各民族高变
区I、高变区n和高变区nl的鉴别能力，鉴别能力均为高变区I>高变
区n>高变区Hl，而且各民族间多态性存在差异。本研究还发现不同
民族的高变区111所在区域不完全相同;同一民族不同地域、不同多态
J性区域的碱基变异也存在差异。在测定的无关个体mtDNA单倍型序列
的基础上做进化距离分析，并构建了各民族内部和民族间的系统发育
树。结论本课题所建立的线粒体DNA序列分析体系是一种对法医疑
难检材进行鉴别的有效、实用的方法，并且本方法已经在实际案件检验
中得到验证。对汉族、黎族、维族、瑶族、藏族群体进行序列分析结果
表明，将检测区域从HVI和HVH扩大到覆盖整个控制区及其周围区域
后大大提高了mtDNA这一遗传标记在法庭科学领域的鉴别能力。同时
在研究过程中检出的新的高变SNP和STR位点也为今后应用mtDNA进
行疑难样本的筛选提供了新的标记。各民族之间多态性比较发现民族间
存在差异，提示应根据不同民族样本建立参照数据库。5个民族之间的
遗传关系分析表明，所选择的群体样本及测序区能够满足群体遗传学分
析的要求，而且新发现的变异位点可以为建立更完善的系统树提供指
标。
Objective In order to improve the discrimination power of mitochondrial DNA in forensic DNA typing, primer pairs which covered the mtDNA control region were designed. The methods of both amplification and DNA sequencing with new primers were established. Polymorphisms of mtDNA control region in 5 ethnic groups in China were analyzed using the established methods to provide a basic database. Molecular evolution and phylogenetic issues were discussed based on the mtDNA database to understand genetic relationship of different ethnic groups in China. Methods The primers were design according to the Anderson's sequence. The amplification system was optimized so that the PCR with different primers can be carried out under the same condition. The PCR products were sequenced using
Sanger's terminator and fluorescent label techniques. Sequences were analyzed and compared base on Sequencing Analysis3.4 and Seq/Ede software. Validation study of forensic science was carried out using forensic samples such as hair, nail and badly degrade bone. The method has also been used in case work to validate the practicability. MAGA2 software was employed to analyze the genetic distance between samples and construct the phylogenentic tree. Result A total of five primer pairs was designed to cover the whole mtDNA control region and the neighbor part. The length of amplicon was from 299 bp to 452bp with different primer pairs. The successful result was obtained even if the DNA template was small to 0.015ng. The mtDNA typing of hair, nail and bone can be carried out with higher successful rate. Sequence analysis revealed that the number of variation site was 269, 316, 141, 117, 159 in Han, Li, Wei, Yao, Zang ethnic group, respectively, while the mean number of base pair with difference was 15.3, 15.9, 12.0, 10.5, 11.3 in Han, Li, Wei, Yao, Zang ethnic group, respectively. The genetic diversity of population samples from Han, Li, Wei, Yao, Zang ethnic group was 1,1, 0.9967, 0.9936, 1, respectively. The result of analysis for discrimination power from three hypervariable regions showed I higher than II and II higher than III. The third hypervariable region was different among the ethnic groups we studied. The analysis of variation in Han group revealed geographic difference. The genetic distances based on the sequence of mtDNA haplotype
have been obtained and the phylogenetic trees were constructed. Conclusion The mtDNA sequencing system using new primers was useful for forensic purpose. For difficult samples, DNA typing with this system was especially available and practical, and has been validated in criminal cases. After extending the sequenced region tb whole control region, the sequence in Han, Li, Wei, Yao, Zang ethnic groups revealed that the discrimination power of mtDNA was increased greatly. The novel STR and SNP loci found in the region of mtDNA also provide useful markers for screening test of forensic samples. The different polymorphisms among ethnic groups imply that it is necessary to establish reference database based on each ethnic group. The study of genetic relationship showed that the selected samples and the sequenced mtDNA region satisfied the request of the genetic analysis. The novel variable site provided some index to establish a perfect phylogenetic tree.
引文
1. Higuchi R, von Beroldingen CH, Sensabaugh GF, et al. DNA typing from single hairs. Nature 1988 Apr 7;332(6164):543-546
2. Koyama H, Iwasa M, Ohtani S, et al. Personal identification from human remains by mitochondrial DNA sequencing. Am J Forensic Med Pathol 2002 Sep;23(3):272-276
3. Lorente JA, Entrala C, Alvarez JC, et al. Social benefits of non-criminal genetic databases: missing persons and human remains identification. Int J Legal Med 2002 Jun;116(3):187-190
4. Montiel R, Malgosa A, Francalacci P. Authenticating ancient human mitochondrial DNA. Hum Biol 2001 Oct; 73(5):689-713
5. Budowle B, Allard MW, Fisher CL, et al. HVⅠ and HVⅡ mitochondrial DNA data in Apaches and Navajos. Int J Legal Med 2002 Aug; 116(4):212-214.
6. Imaizumi K, Parsons TJ, Yoshino M, et al. A new database of mitochondrial DNA hypervariable regions Ⅰ and Ⅱ sequences from 162 Japanese individuals. Int J Legal Med 2002 Apr;116(2):68-73
7. Koyama H, Iwasa M, Maeno Y, et al. Mitochondrial sequence haplotype in tile Japanese population. Forensic Sci Int 2002 Jan 24;125(I):93-96
8. Tsai LC, Lin CY, Lee JC, et al. Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population. Forensic Sci Int 2001 Jun 15;119(2):239-247
9. Tagliabracci A, Turchi C, Buscemi L, et al. Polymorphism of the mitochondrial DNA control region in Italians. Int J Legal Med 2001 ; 114(4-5):224-228
10. Cali F, Le Roux MG, D'Anna R, et al. MtDNA control region and RFLP data for Sicily and France. Int J Legal Med
2001; 114(4-5):229-231
11. Crespillo M, Luque JA, Paredes M, et al. Mitochondrial DNA sequences for 118 individuals from northeastern Spain. Int J Legal Med 2000;114(1-2): 130-132
12. Baasner A, Madea B. Sequence Polymorphisms of the Mitochondrial DNA Control Region in 100 German Caucasians. J Forensic Sci. 2001, (46):1343-1347
13．高俊薇，刘雅诚，唐晖等．指甲DNA的STR分型研究．中国法医学杂志．2003，18(1)：25-27
14．刘雅诚，王静，严江伟等．硅珠法提取骨组织DNA．中国法医学杂志．2003，17(1)：38-39
15. http://www-genome.wi.mit. edu/cgi-bin/primer/primer3.cgi/primer3_www.cgi
16. http://ncbi.nlm.nih.gov/BLAST
17. Parsons TJ, Coble MD. Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome. Croat Med J 2001, 42(3):304-9
18．伍新尧主编．高级法医学．郑州大学出版社，2002，228-231
2. Koyama H, Iwasa M, Ohtani S, et al. Personal identification from human remains by mitochondrial DNA sequencing. Am J Forensic Med Pathol 2002 Sep;23(3):272-276
3. Lorente JA, Entrala C, Alvarez JC, et al. Social benefits of non-criminal genetic databases: missing persons and human remains identification. Int J Legal Med 2002 Jun;116(3):187-190
4. Montiel R, Malgosa A, Francalacci P. Authenticating ancient human mitochondrial DNA. Hum Biol 2001 Oct; 73(5):689-713
5. Budowle B, Allard MW, Fisher CL, et al. HVⅠ and HVⅡ mitochondrial DNA data in Apaches and Navajos. Int J Legal Med 2002 Aug; 116(4):212-214.
6. Imaizumi K, Parsons TJ, Yoshino M, et al. A new database of mitochondrial DNA hypervariable regions Ⅰ and Ⅱ sequences from 162 Japanese individuals. Int J Legal Med 2002 Apr;116(2):68-73
7. Koyama H, Iwasa M, Maeno Y, et al. Mitochondrial sequence haplotype in tile Japanese population. Forensic Sci Int 2002 Jan 24;125(I):93-96
8. Tsai LC, Lin CY, Lee JC, et al. Sequence polymorphism of mitochondrial D-loop DNA in the Taiwanese Han population. Forensic Sci Int 2001 Jun 15;119(2):239-247
9. Tagliabracci A, Turchi C, Buscemi L, et al. Polymorphism of the mitochondrial DNA control region in Italians. Int J Legal Med 2001 ; 114(4-5):224-228
10. Cali F, Le Roux MG, D'Anna R, et al. MtDNA control region and RFLP data for Sicily and France. Int J Legal Med
2001; 114(4-5):229-231
11. Crespillo M, Luque JA, Paredes M, et al. Mitochondrial DNA sequences for 118 individuals from northeastern Spain. Int J Legal Med 2000;114(1-2): 130-132
12. Baasner A, Madea B. Sequence Polymorphisms of the Mitochondrial DNA Control Region in 100 German Caucasians. J Forensic Sci. 2001, (46):1343-1347
13．高俊薇，刘雅诚，唐晖等．指甲DNA的STR分型研究．中国法医学杂志．2003，18(1)：25-27
14．刘雅诚，王静，严江伟等．硅珠法提取骨组织DNA．中国法医学杂志．2003，17(1)：38-39
15. http://www-genome.wi.mit. edu/cgi-bin/primer/primer3.cgi/primer3_www.cgi
16. http://ncbi.nlm.nih.gov/BLAST
17. Parsons TJ, Coble MD. Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome. Croat Med J 2001, 42(3):304-9
18．伍新尧主编．高级法医学．郑州大学出版社，2002，228-231