本文選題：Y染色體　切入點：雙等位基因標記　出處：《華中科技大學》2010年碩士論文　論文類型：學位論文

【摘要】：研究背景人類DNA遺傳標記的發(fā)展主要經(jīng)歷了三個階段:第一階段是80年代中后期建立的限制性片段長度多態(tài)性(restriction fragment length polymorphism, RFLP);第二階段是基于PCR技術(shù)的可變數(shù)目串聯(lián)重復序列(variable number of tandem repeats, VNTR)多態(tài)性,主要包括小衛(wèi)星DNA和微衛(wèi)星DNA;第三階段是雙等位基因標記系統(tǒng)(biallelic markers system),主要包括單核苷酸多態(tài)性(single nucleotide polymorphisms, SNPs)和小片段插入/缺失(insertion/deletions, Indels)。其中,雙等位基因標記系統(tǒng)具有分布廣泛、遺傳穩(wěn)定、突變率低以及易于分析等優(yōu)點,因而逐漸成為研究熱點。Y染色體為父系遺傳,除擬常染區(qū)外,絕大部分為特異性非重組區(qū),單倍型保持完整,不易受重組和回復突變的影響,突變率低,遺傳穩(wěn)定。因此Y染色體遺傳標記的研究,不僅在人類遺傳學上具有重要意義,也為某些特定情況下的法醫(yī)學個體識別和親權(quán)鑒定提供了新的手段,如涉及男性子代的親權(quán)鑒定、混合斑男性成分的檢測、無名男尸的身源確定等具有獨特的應用價值。 目的研究Y染色體上26個雙等位基因標記位點的遺傳多態(tài)性,探討其在法醫(yī)學和人類進化研究中的應用價值。 方法查閱文獻及Y-SNP數(shù)據(jù)庫,挑選在東亞人群中多態(tài)性較好的26個Y染色體雙等位基因標記,用Primer 3軟件自行設(shè)計其靶片段擴增引物和單堿基延伸引物,并將26個位點根據(jù)片段大小及引物間相互作用情況組合成三組進行復合擴增檢測。先利用特異性引物擴增包含各SNP位點的靶基因片段,純化后再利用單堿基延伸引物進行各位點等位基因特異性單堿基延伸反應,以此構(gòu)建多個Y染色體雙等位基因標記的熒光復合擴增檢測體系。然后,用構(gòu)建的熒光復合擴增檢測體系對120名武漢漢族男性無關(guān)健康個體進行分型檢測,并用NETWORK4.5.1.6軟件根據(jù)分型檢測結(jié)果構(gòu)建武漢漢族群體的系統(tǒng)發(fā)生網(wǎng)絡(luò)。 結(jié)果⑴成功構(gòu)建了用于檢測26個Y染色體雙等位基因標記的三組熒光復合擴增檢測體系,每一復合體系中,同一位點的兩個不同等位基因顯示為不同顏色的產(chǎn)物峰,不同位點的等位基因之間片段大小不同;⑵所有26個Y染色體雙等位基因標記在武漢漢族群體中均具有遺傳多態(tài)性,120名武漢漢族男性個體中,共檢出26種單倍型,其頻率范圍為0.0083~0.1250,單倍型多樣性為0.9349;⑶構(gòu)建了武漢漢族群體26個Y染色體雙等位基因標記的系統(tǒng)發(fā)生網(wǎng)絡(luò)。 結(jié)論本文構(gòu)建的26個Y染色體雙等位基因標記熒光復合擴增檢測體系具有簡便、高效、準確的特點,具有較高的法醫(yī)學應用價值。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R394

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