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  本文選題：miniSTR + 復合擴增　； 參考：《河北醫(yī)科大學》2009年碩士論文

【摘要】： 目的:目前,高度降解檢材的DNA分型一直是法醫(yī)DNA領域的一大難題,面對命案現(xiàn)場或重大群體性災難事件中的一些高度腐敗降解檢材,應用現(xiàn)有的短串聯(lián)重復序列(short tandom repeats, STR)分型技術往往不能獲得成功分型及明確的分型結果,為案件偵破或失蹤人員的認定帶來困難。針對這一問題,法醫(yī)學家主要從樣本DNA提取及分型技術兩方面進行研究,如:研發(fā)或改進DNA提取技術,盡量獲取大片段的DNA;應用SNP、miniSTR技術對小片段的DNA進行分型等。2001年9.11恐怖事件中miniSTR技術有力地推動了遇難者個人識別工作的進行,2003年Butler等首次提出miniSTR的概念,并正式命名為miniSTR技術。miniSTR技術就是在設計引物時,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樣本檢測可提高個人識別和親權鑒定的檢測成功率。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。目前,美國的AB公司已開發(fā)出商品化的minifiler試劑盒,其中包括D13S317、D7S820、D2S1338、D21S11、D16S539、D18S51、CSF1PO和FGA,以及性別基因Amelogenin,共九個基因座。minifiler試劑盒能夠解決部分高度降解檢材的DNA分型。為了增加系統(tǒng)效能,美國國家標準及技術研究院(National Institute of Standards andTechnology, NIST)研發(fā)了26個CODIS外的miniSTR基因座,其中,D10S1248、D14S1434、D22S1045三個基因座已被歐洲DNA數(shù)據(jù)庫收錄。美國、日本、西班牙、新加坡、韓國等國家也相繼報道了miniSTR D1S1677、D4S2364、D10S1248等基因座的群體遺傳學數(shù)據(jù)及其法醫(yī)學應用價值,而我國相關研究較少且缺乏群體遺傳學數(shù)據(jù)。 為調(diào)查這些基因座在中國人群中的應用價值,開發(fā)國產(chǎn)miniSTR試劑盒,本研究對D10S1248、D2S441、D1S1677和D9S1122、D10S1435、D17S1301六個miniSTR基因座進行中國山東漢族人群群體遺傳學調(diào)查,并構建兩組熒光標記復合擴增體系,探討其法醫(yī)學應用價值,以期為將miniSTR技術應用于法醫(yī)實踐提供參考。 方法:采用二次Chelex-100法提取120份山東漢族無關健康個體全血基因組DNA。本實驗構建了2個復合擴增體系。復合擴增體系1包括D10S1248, D2S441, D1S1677三個miniSTR基因座,其上游引物5’端分別用6-FAM、HEX、TAMRA熒光標記;復合擴增體系2包括D9S1122、D10S1435、D17S1301三個miniSTR基因座,其上游引物5’端分別用6-FAM、HEX、TAMRA熒光標記。復合擴增反應體系為10μl,對反應體系中的引物濃度、Mg2+、Taq聚合酶、退火溫度、循環(huán)次數(shù)等條件進行優(yōu)化,得到最佳反應條件。PCR復合擴增產(chǎn)物在ABI PRISM 3130、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(chǎn)物片段相對大小并進行樣本基因型分型。依據(jù)本實驗室構建的等位基因標準物及測序結果對各產(chǎn)物進行等位基因命名。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已確定親緣關系的父-子-母三聯(lián)體樣本10例,對6個miniSTR基因座進行遺傳穩(wěn)定性的研究。將9947 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PowerPlex16試劑盒進行DNA分型,比較二者分型的成功率;將尸體解剖的皮膚、肌肉組織、腎臟、脾臟放置于37℃,100%濕度的孵箱中模擬降解檢材,在一天、兩天、四天、一周提取DNA,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PowerPlex16試劑盒進行DNA分型,比較二者分型的成功率。 結果: 1、復合擴增體系的建立: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,6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(chǎn)物,對6個基因座都可實現(xiàn)準確分型。6個基因座的擴增片段長度均小于150bp。 2、群體遺傳學調(diào)查結果: 120名山東漢族無關個體中D10S1248、D2S441、D1S1677、D9S1122、D10S1435、D17S1301基因座分別檢出8、7、6、6、8、8個等位基因和20、18、12、17、20、22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0.750,0.775,0.650,0.708,0.750,0.858;多態(tài)性信息含量(PIC)分別依次為0.75,0.71,0.60,0.69,0.73,0.77;非父排除率(PE)分別依次為0.510,0.553,0.355,0.441,0.51,0.711;個人識別力(PD)分別依次為0.900,0.881,0.809,0.881,0.893,0.913。六個miniSTR基因座的累積非父排除概率為0.988817,累積個人識別力為0.9999975。 3、遺傳穩(wěn)定性分析結果:對10個已經(jīng)確定親權關系的三聯(lián)體樣本研究表明,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。 4、系統(tǒng)靈敏度研究結果:在DNA含量為0.1ng時,PowerPlex16試劑盒擴增產(chǎn)物產(chǎn)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。 5、降解微量檢材分析:對降解兩個月的血液檢材和在孵箱中模擬降解檢材的人體組織,雖然結果中出現(xiàn)了少量雜峰,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。 6、種屬特異性研究:一些常見動物如:猴、豬、牛、狗、兔、魚未檢測到特異性擴增產(chǎn)物。 結論:D10S1248、D2S441、D1S1677、D9S1122、D10S1435和D17S1301六個miniSTR基因座在山東地區(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。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,為開發(fā)國產(chǎn)化miniSTR分型試劑盒奠定了基礎。
[Abstract]:Objective: at present, the DNA classification of highly degraded materials has been a major problem in the field of forensic DNA. In the face of some highly corrupted degradation materials in the scene of life or major mass disaster events, the application of the existing short tandem repeat (short tandom repeats, STR) typing technique often fails to achieve successful typing and clear typing results. For this problem, forensic scientists mainly study from two aspects of sample DNA extraction and typing technology, such as: R & D or improvement of DNA extraction technology, as far as possible to obtain large fragments of DNA; SNP, miniSTR technology for small fragments of DNA in the.2001 years of terror miniSTR The technology has greatly promoted the personal identification work of the victims. In 2003, Butler and so on first proposed the concept of miniSTR and formally named miniSTR technology.MiniSTR technology to shorten the length of the amplified fragment as close as possible to the core repeat sequence when the primers were designed, so that the highly degraded DNA sample test could improve personal knowledge. The European DNA profiling group, EDNAP) is defined as a new generation of genetic markers following STR (the European DNA profiling group, EDNAP). Currently, AB companies in the United States have developed commercialized Minifiler kits. And FGA, as well as the sex gene Amelogenin, a total of nine loci.Minifiler kits can solve the DNA classification of partially degraded samples. In order to increase the system efficiency, the National Institute of standards and Technology (National Institute of Standards andTechnology, NIST) developed 26 CODIS miniSTR genes. 434, three D22S1045 loci have been included in the European DNA database. The United States, Japan, Spain, Singapore, Korea and other countries have also reported the genetic data of miniSTR D1S1677, D4S2364, D10S1248 and other genetic bases and their forensic application value, while our research is less and lack of population genetic data.
In order to investigate the application value of these loci in Chinese population, the domestic miniSTR kits were developed. In this study, the six miniSTR loci of D10S1248, D2S441, D1S1677 and D9S1122, D10S1435, D17S1301 were investigated in the population genetics of Shandong Han population in China, and two groups of fluorescent labeling complex amplification systems were constructed to explore the application price of forensic medicine. Value in order to provide reference for applying miniSTR technology to forensic practice.
Methods: two Chelex-100 methods were used to extract the whole blood genome DNA. from 120 unrelated healthy individuals of Shandong Han. The composite amplification system 1 included three miniSTR loci of D10S1248, D2S441 and D1S1677, and the 5 'end of the upstream primers were labeled with 6-FAM, HEX, TAMRA, and 2 including D9S1122, respectively. D10S1435, D17S1301 three miniSTR loci, its upstream primers 5 'end were labeled with 6-FAM, HEX, TAMRA fluorescence respectively. The compound amplification reaction system was 10 mu L, and the primer concentration, Mg2+, Taq polymerase, annealing temperature and cycle times in the reaction system were optimized, and the optimal reaction conditions were obtained in ABI PRISM 3130. The PRISM 310 gene analyzer automatically completed the data collection of electrophoresis and electrophoresis results. The results were calculated using Genemapper 3.2 to calculate the relative size of the amplified product fragments and carry out the genotyping. The allele frequencies of each product were named according to the allelic standard and sequencing results constructed in our laboratory. The genetic stability of the 6 miniSTR loci was studied by the previous paternity test, which had been identified by this forensic identification center, which had identified the parent and parent three body samples of the relatives, and the 9947 DNA samples were diluted into 1ng, 0.5ng, 0.1ng, 0.05ng for sensitivity detection. In addition, the fresh blood was placed in the natural environment in summer. During the week, two weeks, four weeks, and eight weeks of simulated degradation, 6 miniSTR loci fluorescent tagged composite amplification systems and commercial PowerPlex16 kits were used for DNA typing, and the success rate of the two types was compared; the autopsy skin, muscle tissue, kidney, and spleen were placed at 37, 100% humidity incubator. DNA was extracted at one day, two days, four days and one week by simulated degradation test. The 6 miniSTR loci fluorescent tagged composite amplification system and commercialized PowerPlex16 kit were used for DNA typing, and the success rate of the two genotyping was compared.
Result:
1, the establishment of the composite amplification system: This study established two groups of fluorescent multiplex amplification to detect 6 miniSTR genotypes. The typing results showed that 6 miniSTR loci alleles obtained clear genotyping results, non specific type of non specific amplification products, and 6 gene loci. The exact length of the.6 locus can be achieved. The length of the amplified fragments is less than 150bp..
2, the results of population genetics survey: 120 unrelated individuals of Shandong Han, D10S1248, D2S441, D1S1677, D9S1122, D10S1435, and D17S1301 loci detected 8,7,6,6,8,8 alleles and 20,18,12,17,20,22 genotypes respectively. The genotype frequency distribution was conformed to Hardy-Weinberg balance (P0.05) by chi 2 test. Six loci were in Chinese Han population. The observation value of heterozygosity (Ho) was 0.750,0.775,0.650,0.708,0.750,0.858, and the polymorphism information content (PIC) was respectively 0.75,0.71,0.60,0.69,0.73,0.77, and the non parent exclusion rate (PE) was 0.510,0.553,0.355,0.441,0.51,0.711 respectively, and the individual recognition power (PD) division was 0.900,0.881,0.809,0.881,0.893,0.913. six miniSTR genes in turn. The cumulative non parent exclusion probability is 0.988817, and the cumulative personal recognition power is 0.9999975..
3, the results of genetic stability analysis: the study of three associated samples of 10 identified parental relationships showed that the parents' allele could be inherited steadily to the children, consistent with the Mendel's law of genetics.
4, the results of the system sensitivity study: when the content of DNA is 0.1ng, the output of the PowerPlex16 kit is low, and it is not easy to carry out the correct typing, but the miniSTR compound amplification system can also be used to classify the DNA of 0.05ng.
5, the analysis of degrading trace material: the blood samples degraded for two months and the human tissues that simulated degradation in the incubator, although a small number of hybrid peaks were found in the results, there was no interference to the results of the classification, and the complete classification was obtained in the six miniSTR loci, showing that the miniSTR technology was more than the conventional STR kit. Power.
6, species specific research: some common animals such as monkeys, pigs, cattle, dogs, rabbits and fish did not detect specific amplification products.
Conclusion: the six miniSTR loci of D10S1248, D2S441, D1S1677, D9S1122, D10S1435 and D17S1301 have good genetic polymorphism in the Han population in Shandong area, which conforms to the Mendel genetic rule. The results of the two groups of fluorescent labeling complex amplification systems established in this study are accurate, stable and reliable, with a sensitivity of 0.05ng, for the analysis of trace and degradation. The success rate of DNA 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. It can also be used as a useful supplement to the conventional STR kit, improve the system efficiency, and lay the foundation for the development of domestic miniSTR typing kits.

【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】：2009
【分類號】：D919

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