微测序技术检测武汉汉族26个Y染色体双等位基因标记的遗传多态性

发布时间：2018-03-21 01:58

本文选题：Y染色体　切入点：双等位基因标记　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：研究背景人类DNA遗传标记的发展主要经历了三个阶段:第一阶段是80年代中后期建立的限制性片段长度多态性(restriction fragment length polymorphism, RFLP);第二阶段是基于PCR技术的可变数目串联重复序列(variable number of tandem repeats, VNTR)多态性,主要包括小卫星DNA和微卫星DNA;第三阶段是双等位基因标记系统(biallelic markers system),主要包括单核苷酸多态性(single nucleotide polymorphisms, SNPs)和小片段插入/缺失(insertion/deletions, Indels)。其中,双等位基因标记系统具有分布广泛、遗传稳定、突变率低以及易于分析等优点,因而逐渐成为研究热点。Y染色体为父系遗传,除拟常染区外,绝大部分为特异性非重组区,单倍型保持完整,不易受重组和回复突变的影响,突变率低,遗传稳定。因此Y染色体遗传标记的研究,不仅在人类遗传学上具有重要意义,也为某些特定情况下的法医学个体识别和亲权鉴定提供了新的手段,如涉及男性子代的亲权鉴定、混合斑男性成分的检测、无名男尸的身源确定等具有独特的应用价值。目的研究Y染色体上26个双等位基因标记位点的遗传多态性,探讨其在法医学和人类进化研究中的应用价值。方法查阅文献及Y-SNP数据库,挑选在东亚人群中多态性较好的26个Y染色体双等位基因标记,用Primer 3软件自行设计其靶片段扩增引物和单碱基延伸引物,并将26个位点根据片段大小及引物间相互作用情况组合成三组进行复合扩增检测。先利用特异性引物扩增包含各SNP位点的靶基因片段,纯化后再利用单碱基延伸引物进行各位点等位基因特异性单碱基延伸反应,以此构建多个Y染色体双等位基因标记的荧光复合扩增检测体系。然后,用构建的荧光复合扩增检测体系对120名武汉汉族男性无关健康个体进行分型检测,并用NETWORK4.5.1.6软件根据分型检测结果构建武汉汉族群体的系统发生网络。结果⑴成功构建了用于检测26个Y染色体双等位基因标记的三组荧光复合扩增检测体系,每一复合体系中,同一位点的两个不同等位基因显示为不同颜色的产物峰,不同位点的等位基因之间片段大小不同;⑵所有26个Y染色体双等位基因标记在武汉汉族群体中均具有遗传多态性,120名武汉汉族男性个体中,共检出26种单倍型,其频率范围为0.0083~0.1250,单倍型多样性为0.9349;⑶构建了武汉汉族群体26个Y染色体双等位基因标记的系统发生网络。结论本文构建的26个Y染色体双等位基因标记荧光复合扩增检测体系具有简便、高效、准确的特点,具有较高的法医学应用价值。
[Abstract]:Background the development of human DNA genetic markers has undergone three stages: the first stage is restriction fragment length polymorphisms (RFLPs) established in the middle and late period of 80s, and the second stage is the variable number series based on PCR technology. Repeat sequence variable number of tandem repeat (VNTR) polymorphism, The third stage is the biallelic markers system, which mainly includes single nucleotide polymorphisms (SNPs) and small fragment insertions / deletions, among which the double allelic markers systems are widely distributed. Due to the advantages of stable inheritance, low mutation rate and easy analysis, it has gradually become a hot spot in the study of patrilineal inheritance on chromosome Y, with the exception of pseudochromogenic regions, most of which are specific non-recombination regions, and haplotypes remain intact. Not easily affected by recombination and reverse mutation, the mutation rate is low and genetic stability. Therefore, the study of Y chromosome genetic markers is of great significance not only in human genetics, but also in human genetics. It also provides a new method for forensic individual identification and paternity identification under certain circumstances, such as paternity identification involving male offspring, detection of mixed male component, determination of body origin of unidentified male cadaver and so on. Objective to study the genetic polymorphism of 26 double allelic marker loci on Y chromosome and its application value in forensic medicine and human evolution. Methods A total of 26 polymorphic Y chromosome double alleles were selected from literature and Y-SNP database, and their target fragment amplification primers and single base extension primers were designed by Primer 3 software. The 26 loci were combined into three groups according to the fragment size and the interaction between primers. The target gene fragments containing each SNP locus were amplified by specific primers. After purification, single base extension primers were used to carry out allele-specific single-base extension reaction, so as to construct a fluorescent multiplex amplification detection system for multiple Y chromosome double allelic markers. 120 unrelated healthy individuals of Wuhan Han nationality were detected by using the fluorescent multiplex amplification detection system, and the phylogenetic network of Wuhan Han population was constructed by NETWORK4.5.1.6 software. Results 1 three groups of fluorescent multiplex amplification systems were successfully constructed to detect 26 Y chromosome double alleles. In each complex system, two different alleles at the same locus showed different color peaks. The alleles at different loci were different in size. All 26 Y chromosome double alleles had genetic polymorphisms in Wuhan Han population. A total of 26 haplotypes were detected in 120 Wuhan Han male individuals. The frequency range was 0.0083 (0.1250) and the haplotype diversity was 0.9349m3. The phylogenetic network of 26 Y chromosome double alleles in Wuhan Han population was constructed. Conclusion the 26 Y chromosome double allelic marker fluorescent multiplex amplification system is simple, efficient and accurate, and has high forensic application value.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R394

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