法医线粒体DNA分型及中国不同民族多态性研究

发布时间：2018-04-28 16:58

本文选题：线粒体DNA + 序列分析　；参考：《四川大学》2003年博士论文

【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。
[Abstract]:Objective to improve the forensic identification ability of mitochondrial DNA genetic markers, design suitable primers suitable for forensic medicine, overlay amplification primers in mtDNA control area, establish new amplification and sequencing methods, and investigate the polymorphism of multiethnic mtDNA control area with the method established, and provide population inheritance for mtDNA sequence analysis involving different nationalities. Study the basis; use the obtained data to carry out molecular evolution analysis, explore the relationship between different nationalities and the problem of molecular evolution in the same nation. Methods based on the sequence of the mtDNA control area and its surrounding region, a number of pairs of primers are designed to optimize the amplification system so that the amplification conditions can meet the needs of multiple primers at the same time and use the end of Sanger. Sequence analysis and comparison of DNA, Sequencing Analysis3.4 and Seq / Ede software were carried out by end termination method and fluorescence labeling technique. The validity of the method was tested with hair, nail, trace trace and all kinds of old bone samples, and practical cases were used to verify the validity of the method. 446 samples of unrelated individuals of Han, Li, Uygur, Yao and Tibetan were analyzed, and the mtDNA polymorphism of different ethnic groups was investigated. MEGA 2 software was used to analyze the evolution distance of all ethnic data, and the phylogenetic tree of each ethnic group was constructed and the genetic relationship between ethnic groups was discussed. The results were designed to cover the whole population. 5 pairs of primers in the mtDNA control area and the surrounding area make the length of the amplified products between 299bp and 452bp, and the amplification conditions enable the 5 segments to be amplified under the same cycle parameters.
Chinese abstract
Reliable results were obtained. The method can be used to sequence the total DNA of 0.01 SNG cells.
By sequencing and statistical analysis of blood samples from different ethnic groups, the Han nationality, Li nationality, and Victoria
269316141127159 mutations were detected in the Uygur, Yao and Tibetan groups.
The average variance of each ethnic group is 15.3,15.9,12.0,10.5,11.3.
In the body samples, the total variability was 1, l, 0.9967,0.9936 and L. respectively.
The discrimination ability and identification ability of I and N in hypervariable region and NL in hypervariable region are all I hypervariable in hypervariable region.
The N hypervariable region was Hl, and there were differences among different races.
The region of high variability in 111 is not the same; in the same ethnic group, there are different polymorphisms.
The variation of base region in J region also varies. In the unrelated individuals, mtDNA haplotype sequence was detected.
Based on the analysis of evolutionary distance, we constructed the phylogeny of different races.
Conclusion: the mitochondrial DNA sequence analysis system established in this study is a forensic suspect.
An effective and practical method for identifying difficult materials, and this method has been tested in actual cases.
The results of sequence analysis of Han, Li, Uygur, Yao and Tibetan groups were analyzed.
It shows that the detection area is expanded from HVI and HVH to cover the whole control area and its surrounding area.
It greatly enhanced the identification ability of mtDNA as a genetic marker in forensic science.
The new hypervariable SNP and STR loci detected during the study also apply to mtDNA in the future.
The screening of difficult samples provides new markers.
There are differences, suggesting that reference databases should be established based on different ethnic groups,.5 ethnic groups.
Genetic relationship analysis showed that the selected population samples and sequencing regions could meet the genetic credits of population.
Moreover, the newly discovered mutation sites can provide a reference for building a more complete system tree.
Mark.

【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2003
【分类号】：D919

【引证文献】