本文選題：miniSTR + DNA分型　； 參考：《河北醫(yī)科大學》2008年碩士論文

【摘要】： 目的:自上世紀80年代以來,短串聯(lián)重復序列(short tandem repeats, STR)遺傳標記系統(tǒng),由于在人類基因組中分布廣泛,高度的遺傳多態(tài)性以及簡單快捷的檢測方法而成為法庭科學鑒定中最常用、發(fā)展最成熟的技術,被廣泛應用于法醫(yī)學等領域,STR數(shù)據(jù)成為各國法庭科學數(shù)據(jù)庫的主要構成。目前法醫(yī)學主要運用常染色體STR基因座和線粒體DNA遺傳標記,能夠解決大部分個體識別和親權鑒定案例。然而,在許多法醫(yī)案例中,DNA樣本由于各種原因而被高度降解或被混入環(huán)境污染物,在使用商品化STR復合擴增試劑盒進行檢測時,片段較長的STR基因座往往無法進行有效的擴增,片段檢測不到信號,而這個問題隨著PCR反應產生大量的PCR產物而被擴大。miniSTR基因座分析技術在設計引物時,使其盡可能靠近核心重復序列而縮短擴增片段長度,用于高度降解的DNA樣本檢測可提高檢測成功率。miniSTR基因座被歐洲DNA分型工作組(the european DNA profiling group, EDNAP)定義為繼STR之后的新一代遺傳標記。美國、日本、西班牙、新加坡、韓國等國家也相繼報道了miniSTR基因座的群體遺傳學數(shù)據(jù)及其法醫(yī)學應用價值,而我國相關報道較少并缺乏群體遺傳學數(shù)據(jù)。本研究選取D3S3053, D6S474, D20S482, D1GATA113, D2S1776, D4S2408等六個miniSTR基因座,調查其在中國漢族人群中的遺傳多態(tài)性,并探討其法醫(yī)學應用價值。 方法:采用天根血液基因組DNA提取試劑盒提取120份河北漢族無關健康個體全血基因組DNA。將D20S482、D2S1776兩個基因座的上游引物5’端用6-FAM熒光標記,D3S3053、D1GATA113兩個基因座的上游引物5’端用HEX熒光標記,D6S474、D4S2408兩個基因座的上游引物5’端用TAMRA熒光標記,對所有樣本的六個miniSTR基因座進行PCR復合擴增,其中D3S3053、D6S474、D20S482三個基因座進行復合擴增,D1GATA113、D2S1776、D4S2408三個基因座進行復合擴增。復合擴增反應體系為10μl,分別加入各個基因座特異性的引物,并設定適宜的退火溫度,經(jīng)28個循環(huán)后,可得到各個基因座長度不等的等位基因片段。PCR復合擴增產物在ABI PRISM 310基因分析儀上自動完成進樣電泳和電泳結果數(shù)據(jù)收集,結果用Genemapper 3.2計算擴增產物片段相對大小并進行樣本基因型分型。計算各基因座基因頻率及各法醫(yī)學參數(shù)。選取本法醫(yī)鑒定中心既往親子鑒定的家系樣本9例,對6個miniSTR基因座進行遺傳穩(wěn)定性的研究。提取同一尸體的心臟、肝臟、脾臟、肺組織、腎臟、腦組織、肌肉組織、皮膚及尸體血痕檢材進行組織同一性分析。將9947A DNA樣本稀釋成1ng、0.5ng、0.1ng、0.05ng進行靈敏度檢測。此外,將新鮮血液放置于夏季的自然環(huán)境中一周、兩周、四周和八周模擬降解檢材,應用本研究構建的6個miniSTR基因座熒光標記復合擴增分型體系及商品化常規(guī)STR試劑盒進行DNA分型,比較二者分型的成功率。 結果:本研究建立了兩組熒光復合擴增檢測6個miniSTR基因座基因型的方法。復合擴增檢測樣本的分型結果顯示,六個miniSTR基因座等位基因都獲得了清晰的基因型分型結果,無干擾分型的非特異性擴增產物,對6個基因座都可實現(xiàn)準確分型。相關遺傳學參數(shù):六個基因座的擴增片段長度均小于150bp。120名河北漢族無關個體中D3S3053、D6S474、D20S482、D1GATA113、D2S1776、D4S2408基因座分別檢出6、6、7、8、8、7個等位基因和14、16、15、27、27、23種基因型,基因型頻率分布經(jīng)χ2檢驗均符合Hardy-Weinberg平衡(P0.05)。六個基因座在中國漢族人群的雜合度觀察值(Ho)分別為0.652,0.758,0.758,0.667,0.617,0.85;多態(tài)性信息含量(PIC)分別依次為0.64,0.68,0.69,0.83,0.85,0.83;非父排除率(PE)分別依次為0.298,0.523,0.523,0.313,0.271,0.662;個人識別力(PD)分別依次為0.862,0.857,0.877,0.943,0.947,0.917。六個miniSTR基因座的累積非父排除概率為0.97,累積個人識別力為0.9999994。遺傳穩(wěn)定性分析:對9個已經(jīng)確定親權關系的家系樣本研究表明,父母的等位基因能夠穩(wěn)定地遺傳給子女,符合孟德爾遺傳定律。組織同一性分析:對來自同一尸體不同組織的檢測表明,六個基因座在同一個體中具有同一性,符合STR基因座用做法醫(yī)DNA分析的標準。系統(tǒng)靈敏度研究:在DNA含量為0.1ng時, Identifile試劑盒擴增產物產量比較低,不易進行正確的分型,而應用miniSTR復合擴增系統(tǒng)對0.05ng的DNA還可以進行分型。降解微量檢材研究:對降解兩個月的檢材,雖然擴增的特異度有所下降,但對分型結果沒有干擾,在六個miniSTR基因座中均得到了完整分型,顯示了miniSTR技術比常規(guī)STR試劑盒具有更高的分型成功率。 結論:D3S3053、D6S474、D20S482、D1GATA113、D2S1776和D4S2408六個miniSTR基因座在河北地區(qū)漢族群體中具有較好的遺傳多態(tài)性,符合孟德爾遺傳規(guī)律,具有組織同一性。本研究建立的兩組熒光標記復合擴增體系分型結果準確、穩(wěn)定可靠,靈敏度達0.05ng,對于分析微量、降解DNA的成功率較常規(guī)STR試劑盒明顯升高,可用于法醫(yī)學親權鑒定與個人識別,并在高度降解檢材的檢測中具有較高的應用價值,而且可作為常規(guī)STR試劑盒的有益補充,提高系統(tǒng)效能。
[Abstract]:Objective: since 80s, the short tandem repeats (STR) genetic marker system has been widely used in forensic science and other fields because of its wide distribution, high genetic polymorphism and simple and fast detection methods in the human genome. STR data has become the main component of the National Forensic Science Database. Currently, forensic medicine mainly uses the autosomal STR loci and mitochondrial DNA genetic markers to solve most individual identification and paternity cases. However, in many forensic cases, DNA samples are highly degraded or mixed into environmental pollutants for a variety of reasons. When the commercialized STR complex amplification kit was used to detect the longer STR loci, the fragment was often unable to be amplified effectively, and the fragment was not detected. The problem was enlarged with the PCR reaction to produce a large number of PCR products, and the.MiniSTR gene pedestal analysis technique was expanded as close as possible to the core repeat sequence when the primers were designed. Shortening the length of the amplified fragment, for highly degraded DNA samples, can improve the detection success rate of the.MiniSTR loci by the European DNA typing group (the European DNA profiling group, EDNAP) as a new generation of genetic markers following STR. The United States, Japan, Spain, Singapore, and South Korea have also reported the miniSTR gene. The population genetics data and the application value of forensic medicine are less and lack of population genetics data in our country. This study selected six miniSTR loci, such as D3S3053, D6S474, D20S482, D1GATA113, D2S1776, D4S2408 and so on, to investigate the genetic polymorphism of the Chinese Han population and to explore the application value of their forensic medicine.
Methods: the genomic DNA extraction kit was used to extract 120 copies of the whole blood genome DNA. of unrelated healthy individuals from Hebei Han. The upstream primers of D20S482, D2S1776 two loci were labeled with 6-FAM fluorescence, D3S3053, D1GATA113 two loci were labeled with HEX fluorescence, D6S474, D4S2408 two loci. The 5 'end of the primer was labeled with TAMRA fluorescence, and the six miniSTR loci of all the samples were amplified by PCR, in which three loci of D3S3053, D6S474, D20S482 were amplified, and three loci of D1GATA113, D2S1776 and D4S2408 were multiplex amplified. The compound amplification reaction body was 10 micron L, and the specific primers were added to each loci, respectively. And the suitable annealing temperature was set. After 28 cycles, the.PCR composite amplified products of different allele length could be obtained on the ABI PRISM 310 gene analyzer. The results were obtained by using Genemapper 3.2 to calculate the relative size of the amplified product fragments and carry out the sample genotypes. The genetic stability of the 6 miniSTR loci was studied in 9 family samples of the previous paternity test in the forensic identification center. The samples of the same body were extracted from the heart, liver, spleen, lung tissue, kidney, brain tissue, muscle tissue, skin and corpse blood stains. 9947A DNA samples were diluted into 1ng, 0.5ng, 0.1ng, 0.05ng for sensitivity detection. In addition, the fresh blood was placed in the summer natural environment for a week, two weeks, four weeks and eight weeks, and 6 miniSTR loci fluorescent tagged composite amplification systems and commercial conventional STR reagents were constructed. The box is typed by DNA, and the success rate of the two types is compared.
Results: two groups of fluorescent multiplex amplification were established to detect the genotypes of 6 miniSTR loci. The typing results showed that six miniSTR loci alleles obtained clear genotyping results without interference type non specific amplification products, and all 6 loci could be accurately divided. Related genetic parameters: the length of the amplified fragment of the six loci was less than the D3S3053, D6S474, D20S482, D1GATA113, D2S1776, and D4S2408 loci in the unrelated individuals of the Hebei Han people, and the D4S2408 loci detected the 6,6,7,8,8,7 alleles and the 14,16,15,27,27,23 genotypes respectively. The frequency distribution of the basic genotype was all in line with the Hardy-Weinberg level by the x 2 test. P0.05. The observation value of heterozygosity (Ho) of the six loci in Chinese Han population (Ho) was 0.652,0.758,0.758,0.667,0.617,0.85, and the polymorphism information content (PIC) was respectively 0.64,0.68,0.69,0.83,0.85,0.83, and the non parent exclusion rate (PE) was respectively 0.298,0.523,0.523,0.313,0.271,0.662, and the individual recognition power (PD) in turn was 0.862,0., respectively. The cumulative non paternal exclusion probability of 857,0.877,0.943,0.947,0.917. six miniSTR loci is 0.97, and the cumulative individual recognition power is 0.9999994. genetic stability analysis. The family sample study of 9 identified parental relationships shows that the parents' alleles can be inherited stably to the daughter and conform to Mendel's law of inheritance. Analysis: the detection of different tissues from the same body showed that the six loci had the same character in the same individual, which accords with the standard of the STR loci for forensic DNA analysis. System sensitivity study: when the DNA content is 0.1ng, the production of Identifile kit is low, and the correct typing is not easy, and the miniSTR compound expansion is used. The increasing system can also be classified for the DNA of 0.05ng. Degradation of trace material for two months degradation, although the specificity of the amplification has declined, but there is no interference to the results of the classification, and the complete typing in the six miniSTR loci shows that the miniSTR technology has a higher classification success rate than the conventional STR kit.
Conclusion: the six miniSTR loci of D3S3053, D6S474, D20S482, D1GATA113, D2S1776 and D4S2408 have good genetic polymorphisms in the Han population in Hebei region, which conform to the genetic regularity of Mendel and have the identity of tissue. The results of the two groups of fluorescent labeled compound amplification in this study are accurate, stable and reliable, and the sensitivity is 0.05ng, and In the analysis of trace amounts, the success rate of DNA degradation is significantly higher than that of the conventional STR kit. It can be used in forensic paternity identification and personal identification, and has high application value in the detection of highly degraded materials, and can be used as a useful supplement to the conventional STR kit and improve the efficiency of the system.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：D919

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