本文選題：線粒體DNA + 序列分析　； 參考：《四川大學(xué)》2003年博士論文

【摘要】： 目的 為了提高線粒體DNA這一遺傳標(biāo)記的法醫(yī)學(xué)鑒別能力，設(shè)計(jì)適合法醫(yī)學(xué)檢材的、覆蓋mtDNA控制區(qū)的擴(kuò)增引物，建立新的擴(kuò)增與測序方法；應(yīng)用所建立的方法調(diào)查多民族mtDNA控制區(qū)多態(tài)性，為涉及不同民族的mtDNA序列分析提供群體遺傳學(xué)基礎(chǔ)；應(yīng)用獲得的數(shù)據(jù)進(jìn)行分子進(jìn)化分析，探討各民族間的親緣關(guān)系及同一民族內(nèi)的分子進(jìn)化問題。方法 根據(jù)mtDNA控制區(qū)及其周圍區(qū)域的序列，設(shè)計(jì)多對引物，探索優(yōu)化擴(kuò)增體系，使擴(kuò)增條件能夠同時滿足多對引物的需要，用Sanger末端終止法及熒光標(biāo)記技術(shù)對樣本進(jìn)行DNA測序，Sequencing Analysis3.4和Seq／Ede軟件進(jìn)行序列分析和比對。用毛發(fā)、指甲、微量血痕及各種陳舊骨骼樣本對所建立的方法進(jìn)行法醫(yī)學(xué)有效性測試，并用實(shí)際案例驗(yàn)證其實(shí)用性；應(yīng)用所建立方法對漢族、黎族、維吾爾族、瑤族、藏族無關(guān)個體樣本共446份進(jìn)行序列分析，調(diào)查不同民族mtDNA多態(tài)性；應(yīng)用MEGA 2軟件對所得各民族數(shù)據(jù)做進(jìn)化距離分析，，并構(gòu)建各民族內(nèi)部和民族間的系統(tǒng)發(fā)育樹，探討各民族間的遺傳關(guān)系。結(jié)果 設(shè)計(jì)了覆蓋整個mtDNA控制區(qū)及周圍區(qū)域的5對引物，使各段擴(kuò)增產(chǎn)物長度在299bp到452bp之間，統(tǒng)一了擴(kuò)增條件使5段序列可以在相同循環(huán)參數(shù)下擴(kuò)增。對陳舊骨骼、毛發(fā)、指甲等法醫(yī)學(xué)樣本均 中文摘要 得到可靠結(jié)果。該方法可以對細(xì)胞總DNA為0.01 sng的樣本進(jìn)行測序。 通過對不同民族血樣的序列測定和統(tǒng)計(jì)學(xué)分析，分別在漢族、黎族、維 吾爾族、瑤族、藏族群體中各檢出269、316、141、127、159個突變位 點(diǎn);各民族平均變異數(shù)分別為15.3、15.9、12.0、10.5、11.3，在所用群 體樣本中，總變異度分別為1、l、0.9967、0.9936、l。分析各民族高變 區(qū)I、高變區(qū)n和高變區(qū)nl的鑒別能力，鑒別能力均為高變區(qū)I高變 區(qū)n高變區(qū)Hl，而且各民族間多態(tài)性存在差異。本研究還發(fā)現(xiàn)不同 民族的高變區(qū)111所在區(qū)域不完全相同;同一民族不同地域、不同多態(tài) J性區(qū)域的堿基變異也存在差異。在測定的無關(guān)個體mtDNA單倍型序列 的基礎(chǔ)上做進(jìn)化距離分析，并構(gòu)建了各民族內(nèi)部和民族間的系統(tǒng)發(fā)育 樹。結(jié)論本課題所建立的線粒體DNA序列分析體系是一種對法醫(yī)疑 難檢材進(jìn)行鑒別的有效、實(shí)用的方法，并且本方法已經(jīng)在實(shí)際案件檢驗(yàn) 中得到驗(yàn)證。對漢族、黎族、維族、瑤族、藏族群體進(jìn)行序列分析結(jié)果 表明，將檢測區(qū)域從HVI和HVH擴(kuò)大到覆蓋整個控制區(qū)及其周圍區(qū)域 后大大提高了mtDNA這一遺傳標(biāo)記在法庭科學(xué)領(lǐng)域的鑒別能力。同時 在研究過程中檢出的新的高變SNP和STR位點(diǎn)也為今后應(yīng)用mtDNA進(jìn) 行疑難樣本的篩選提供了新的標(biāo)記。各民族之間多態(tài)性比較發(fā)現(xiàn)民族間 存在差異，提示應(yīng)根據(jù)不同民族樣本建立參照數(shù)據(jù)庫。5個民族之間的 遺傳關(guān)系分析表明，所選擇的群體樣本及測序區(qū)能夠滿足群體遺傳學(xué)分 析的要求，而且新發(fā)現(xiàn)的變異位點(diǎn)可以為建立更完善的系統(tǒng)樹提供指 標(biāo)。
[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.

【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2003
【分類號】：D919

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 蔡繼峰,廖志鋼;嗜尸性蠅類線粒體DNA分子標(biāo)記檢測的研究進(jìn)展[J];法醫(yī)學(xué)雜志;2005年01期

相關(guān)碩士學(xué)位論文 前2條

1 荊玉婷;線粒體DNA單核苷酸多態(tài)性（SNPs）微測序檢測方法的研究[D];山西醫(yī)科大學(xué);2007年

2 熊楓;應(yīng)用細(xì)胞色素氧化酶亞基I鑒定蠅科常見嗜尸性蠅種[D];中南大學(xué);2012年

本文編號：1816114