發(fā)布時(shí)間：2018-06-27 11:45

  本文選題：miniSTR + non-CODIS基因座�。� 參考：《河北醫(yī)科大學(xué)》2009年博士論文

【摘要】： 目的:在法醫(yī)DNA檢驗(yàn)中,STR技術(shù)已經(jīng)廣泛用于個(gè)人識別和親權(quán)鑒定方面。但是法醫(yī)檢案工作中,常常由于高溫、潮濕、暴曬、微生物等因素的影響,使檢材中的DNA分子被損壞,發(fā)生斷裂,分子變小,大片段丟失。在這些情況下應(yīng)用STR技術(shù)不能成功地得出結(jié)論,經(jīng)常會(huì)出現(xiàn)“優(yōu)勢擴(kuò)增”或者“無效擴(kuò)增”,這就給正確的DNA分型帶來困難。重新設(shè)計(jì)引物,減小擴(kuò)增產(chǎn)物片段長度是目前解決DNA高度降解檢材檢驗(yàn)問題的一種新的方法。2001年9·11恐怖事件時(shí)這種方法被應(yīng)用于遇難者的尸源認(rèn)定,同時(shí)被正式命名為miniSTR技術(shù)。目前,AB公司已經(jīng)開發(fā)出針對CODIS系統(tǒng)以內(nèi)8個(gè)基因座的miniSTR商品化試劑盒。但由于CODIS系統(tǒng)內(nèi)一些基因座側(cè)翼序列不適合設(shè)計(jì)新的引物,或者等位基因范圍太大,使這些基因座的擴(kuò)增片段長度難以縮短到理想長度(125bp),因而并非CODIS系統(tǒng)內(nèi)所有的STR基因座都適用于設(shè)計(jì)成miniSTR基因座。為解決這個(gè)問題,增加系統(tǒng)效能,非常有必要尋找CODIS系統(tǒng)之外的更多的miniSTR基因座,并進(jìn)行人群調(diào)查。因此,本實(shí)驗(yàn)的目的是在數(shù)據(jù)庫中篩選出適合中國漢族人群特征的miniSTR基因座,然后將其構(gòu)建成新的miniSTR熒光標(biāo)記復(fù)合擴(kuò)增體系;并按照美國DNA分析方法技術(shù)工作組(TWGDAM)的指導(dǎo)方案,對復(fù)合擴(kuò)增系統(tǒng)的靈敏度,基因座的種屬特異性,案例應(yīng)用,降解模型,陳舊樣本等法醫(yī)學(xué)應(yīng)用進(jìn)行初步研究。最終構(gòu)建成符合中國漢族人群特征的miniSTR復(fù)合擴(kuò)增體系,從而推動(dòng)法醫(yī)DNA試劑盒的國產(chǎn)化進(jìn)程。 方法:①參考Coble等的引物,在中國河北漢族120名無關(guān)個(gè)體中進(jìn)行26個(gè)miniSTR基因座PCR擴(kuò)增,獲得河北漢族群體遺傳學(xué)數(shù)據(jù),根據(jù)數(shù)據(jù)初步篩選出8個(gè)多態(tài)性較高、片段較短的miniSTR基因座;在中國其他地區(qū)754個(gè)無關(guān)個(gè)體中進(jìn)行這8個(gè)miniSTR基因座PCR擴(kuò)增,擴(kuò)增產(chǎn)物使用聚丙烯酞胺凝膠電泳分離,銀染法顯色,獲得8個(gè)基因座的群體遺傳學(xué)數(shù)據(jù)和各基因座的常見等位基因。②采用國產(chǎn)DNA聚合酶構(gòu)建這8個(gè)miniSTR基因座的復(fù)合擴(kuò)增體系,利用熒光復(fù)合擴(kuò)增技術(shù)擴(kuò)增,ABI310/3130基因分析儀對產(chǎn)物進(jìn)行檢測,Genemapper3.2軟件分析結(jié)果;根據(jù)不同引物濃度、Mg~(2+)濃度、不同退火溫度、不同循環(huán)次數(shù)下復(fù)合擴(kuò)增的效果對復(fù)合體系進(jìn)行優(yōu)化。③根據(jù)群體遺傳學(xué)研究的結(jié)果,應(yīng)用分子克隆技術(shù)構(gòu)建各基因座等位基因標(biāo)準(zhǔn)分型物,并根據(jù)國際法醫(yī)遺傳學(xué)會(huì)(international society of forensic genetics, ISFG )推薦的命名原則對各等位基因進(jìn)行命名,同時(shí)根據(jù)檢測數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析編制出miniSTR復(fù)合擴(kuò)增體系的基因分型模版。④對該熒光標(biāo)記復(fù)合擴(kuò)增體系的靈敏度、種屬特異性、可重復(fù)性及對陳舊、腐敗降解檢材DNA的檢測能力進(jìn)行了研究;用實(shí)際案例進(jìn)行驗(yàn)證,并與商品化試劑盒進(jìn)行比較。 結(jié)果:①成功構(gòu)建了兩個(gè)熒光標(biāo)記miniSTR復(fù)合擴(kuò)增體系,其中復(fù)合體系1包含D10S1248, D2S441, D1S1677和D9S2157四個(gè)基因座,復(fù)合體系2包含D9S1122, D10S1435, D12ATA63, D2S1776和Amelogenin五個(gè)基因座。應(yīng)用該體系對300份中國漢族無關(guān)個(gè)體樣本進(jìn)行檢驗(yàn)分析,經(jīng)群體遺傳學(xué)計(jì)算,該系統(tǒng)的累積個(gè)人識別率和非父排除率分別為0.99999999228和0.98547130732。②本實(shí)驗(yàn)構(gòu)建的兩個(gè)復(fù)合擴(kuò)增體系條件易于優(yōu)化,采用國產(chǎn)Taq酶進(jìn)行擴(kuò)增,即可得到滿意的擴(kuò)增產(chǎn)物和分型結(jié)果。相對于國外昂貴的試劑盒,它是一種成本低廉但效率高的miniSTR基因座復(fù)合擴(kuò)增體系。③應(yīng)用分子克隆技術(shù),對9個(gè)基因座的64個(gè)等位基因進(jìn)行克隆,經(jīng)過調(diào)整各基因座等位基因的含量,構(gòu)建出了兩組復(fù)合體系的等位基因標(biāo)準(zhǔn)對照體系。根據(jù)實(shí)驗(yàn)數(shù)據(jù)和測序的命名結(jié)果設(shè)置基因分析命名參數(shù)指標(biāo),建立了兩個(gè)熒光標(biāo)記miniSTR復(fù)合擴(kuò)增體系的基因分型命名模版,可以對樣本準(zhǔn)確分型。④法醫(yī)應(yīng)用性研究證明該體系具有良好的種屬特異性,除恒河猴在Amelogenin出現(xiàn)色譜峰外,其余8個(gè)基因座在8種常見動(dòng)物中均無特異性產(chǎn)物峰出現(xiàn);對同一個(gè)體的不同組織檢測結(jié)果一致;在0.125ngDNA模板量的情況下,可對9個(gè)基因座進(jìn)行正確分型;通過對陳舊樣本和降解模型的研究證明,該體系對降解檢材有良好的擴(kuò)增效果,較常規(guī)大片段STR試劑盒擴(kuò)增成功率高,可用于常規(guī)試劑盒擴(kuò)增失敗的降解樣本的分型;通過8個(gè)實(shí)際案例證明,該體系能用于法醫(yī)學(xué)實(shí)踐中,結(jié)果與鑒定結(jié)論一致。 結(jié)論:本實(shí)驗(yàn)成功的構(gòu)建了適合中國漢族人群的兩個(gè)熒光標(biāo)記miniSTR復(fù)合擴(kuò)增體系。該熒光標(biāo)記復(fù)合擴(kuò)增體系可應(yīng)用于ABI 310/3130檢測平臺,該體系擴(kuò)增條件易于優(yōu)化,成本低廉,分型結(jié)果穩(wěn)定,種屬特異性好,靈敏度和準(zhǔn)確度高,對降解檢材擴(kuò)增成功率高,可以應(yīng)用于法醫(yī)學(xué)實(shí)際檢案中,特別適合對陳舊檢材和降解檢材的DNA檢測。為國內(nèi)法醫(yī)DNA分析提供了一個(gè)成本低,性能好的熒光標(biāo)記miniSTR復(fù)合擴(kuò)增檢驗(yàn)系統(tǒng),具有重大的應(yīng)用價(jià)值。
[Abstract]:Objective: in forensic DNA test, STR technology has been widely used in personal identification and paternity identification. However, in the work of forensic examination, the effects of high temperature, damp, insolation, microorganism and other factors make the DNA molecules in the material damaged, broken, small, and lost. In these cases, the application of STR technology can not be used. The conclusion is that "advantage amplification" or "ineffective amplification" often occur, which makes the correct DNA typing difficult. Redesigning primers to reduce the length of the amplified fragment is a new method to solve the problem of DNA highly degrading test. This method was applied to the victims of the 9. 9. 11 terrorist incident. At present, AB has developed a commercialized kits for 8 loci within the CODIS system. But because some of the loci sequences in the CODIS system are not suitable for the design of new primers, or the range of alleles is too large, the length of the amplified fragment of these loci is difficult. To reduce to the ideal length (125bp), all STR loci in the CODIS system are not suitable for the design of miniSTR loci. In order to solve this problem and increase the system efficiency, it is very necessary to find more miniSTR loci outside the CODIS system and conduct a crowd investigation. Therefore, the purpose of this experiment is to screen out the database in the database. The miniSTR loci suitable for the characteristics of the Chinese Han population were constructed and constructed into a new miniSTR fluorescent labeling complex amplification system, and the sensitivity of the multiplex amplification system, the species specificity of the loci, the case application, the degradation model, the old sample and other forensic medicine should be established according to the guidance scheme of the American DNA analysis method technical working group (TWGDAM). A preliminary study was conducted to construct a miniSTR multiplex system that is consistent with the characteristics of the Chinese Han population, thereby promoting the localization process of the forensic DNA kit.
Methods: (1) based on the primers of Coble and other primers, 26 miniSTR loci were amplified by PCR in 120 unrelated individuals of Hebei Han, and the genetic data of Hebei Han population were obtained. According to the data, 8 polymorphic and short miniSTR loci were screened out, and the 8 miniSTR bases were carried out among 754 unrelated individuals in other regions of China. The amplified products were separated by phthalamine polyacrylamide gel electrophoresis with PCR amplification. The genetic data of 8 loci and the common alleles of each loci were obtained by silver staining. The composite amplification system of the 8 miniSTR loci was constructed by homemade DNA polymerase and amplified by fluorescence combined amplification and ABI310/3130 genotypes. Analysis of the product, Genemapper3.2 software analysis results, according to the concentration of primers, Mg~ (2+) concentration, different annealing temperatures, different cycles of multiplex amplification effect on the composite system optimization. Thirdly, according to the results of population genetics research, the application of sub cloning technology to construct the standard genotypes of the alleles of each gene pedestal, And according to the named principle recommended by the international society of forensic genetics (ISFG), the allele of each allele was named, and the genotyping template of the miniSTR composite amplification system was compiled according to the statistical analysis of the detected data. Sex, repeatability, and detection ability of old and corrupt degradation materials DNA were studied, verified by actual cases, and compared with commercialized kits.
Results: (1) two fluorescent labeled miniSTR composite amplification systems were successfully constructed, in which the compound system 1 contains four loci of D10S1248, D2S441, D1S1677 and D9S2157, and the compound system 2 contains five loci of D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin. According to the population genetic calculation, the cumulative individual recognition rate and the non parent exclusion rate of the system are 0.99999999228 and 0.98547130732., respectively, and the two complex amplification system conditions constructed by this experiment are easy to be optimized. The amplified products and the typing results can be obtained with the homemade Taq enzyme. It is a compound amplification system of miniSTR loci with low cost but high efficiency. (3) using molecular cloning technology, cloning 64 alleles of 9 loci and adjusting the content of alleles of each gene base, a standard control system of alleles for two groups of alleles is constructed. Results by setting the parameter index of gene analysis and naming, two genotyping and naming templates of the miniSTR multiplex amplification system were established, and the samples can be classified accurately. 4. Forensic application studies show that the system has good species specificity, except that the other 8 loci are common in 8 kinds except for the Amelogenin peak of Ganges RIver monkey in the present chromatographic peak. No specific product peak appeared in animals; the results of detection of different tissues of the same individual were consistent, and 9 loci could be properly typed in the case of 0.125ngDNA template. Through the study of old samples and degradation models, it was proved that the system had good amplification effect on degrading test material, and was amplified by the conventional large fragment STR kit. The success rate is high and can be used for the classification of the failure samples of the conventional kit amplification failure. Through 8 practical cases, it is proved that the system can be used in forensic practice, and the result is consistent with the identification conclusion.
Conclusion: this experiment successfully constructed two fluorescent labeled miniSTR multiplex amplification systems suitable for the Chinese Han population. The fluorescent labeling complex amplification system can be applied to the ABI 310/3130 detection platform. The amplification conditions are easy to be optimized, the cost is low, the result is stable, the species specificity is good, the sensitivity and accuracy are high, and the degradation detection is high. The success rate of material amplification is high. It can be applied to the actual forensic examination case, especially suitable for the DNA detection of old material and degrading test material. It provides a low cost and good performance fluorescence labeled miniSTR composite amplification test system for the DNA analysis of domestic forensic medicine, which has a significant value in application.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：D919

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