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  本文選題：STR　切入點(diǎn)：miniSTR　出處：《四川大學(xué)》2007年博士論文

【摘要】： 目的為了提高分析DNA高度降解樣本的成功率，探索一些針對高度降解DNA分析的新方法、新指標(biāo)十分必要。本課題旨在構(gòu)建一些有利于分析高度降解DNA的方法，以彌補(bǔ)商品化STR復(fù)合擴(kuò)增試劑盒在分析高度降解DNA的不足，增加法醫(yī)學(xué)個人識別能力。 方法從法醫(yī)檢案的實(shí)踐中挑選出商品化STR復(fù)合擴(kuò)增試劑盒DNA分析成功率最低的4個STR基因座，通過修改引物結(jié)合區(qū)的位置，減小擴(kuò)增片段的長度，將其構(gòu)建一個復(fù)合擴(kuò)增熒光檢測和自動化分型體系(miniSTR)。將miniSTR與商品化STR復(fù)合擴(kuò)增試劑盒的分型結(jié)果進(jìn)行一致性、抗降解和抗PCR抑制物能力比對實(shí)驗(yàn)，依據(jù)DNA分析技術(shù)工作組(TWGDAM)指南進(jìn)行法醫(yī)學(xué)可行性研究。 選擇5個單核苷酸多態(tài)性基因座(SNP)作為研究對象，3個(rs999842、rs922992、rs924181)定位于常染色體，一個(rs997262)定位于X染色體，一個(m9)位于Y染色體。將其構(gòu)建成一個超短片段長度PCR復(fù)合擴(kuò)增體系，利用多重單堿基延伸熒光檢測方法進(jìn)行分型，與構(gòu)建的miniSTR進(jìn)行抗降解和抗PCR抑制劑能力對比實(shí)驗(yàn)，并進(jìn)行法醫(yī)學(xué)可行性、群體遺傳學(xué)和性別判斷的研究。 構(gòu)建一個PCR內(nèi)對照物，使其能與商品化STR復(fù)合擴(kuò)增試劑盒一同擴(kuò)增，一同電泳，一同自動化分型。通過觀察內(nèi)對照產(chǎn)物峰高情況，確定PCR反應(yīng)中抑制物的存在和作用情況。結(jié)合案例進(jìn)行法醫(yī)學(xué)可行性評估。 結(jié)果通過統(tǒng)計(jì)220份法醫(yī)生物物證樣本的DNA分型結(jié)果，發(fā)現(xiàn)商品化STR復(fù)合擴(kuò)增試劑盒對D7S820、D18S51、CSF1P0、D2S1338、FGA五個基因座分型的成功率最低。由于FGA的核心重復(fù)基序的序列太長，無法將其擴(kuò)增片段長度減小到理想的程度，不適合進(jìn)行小片段STR設(shè)計(jì)。于是我們建立了一個含有D7S820、D18S51、CSF1P0和D2S1338共4個基因座的復(fù)合擴(kuò)增熒光檢測體系(miniSTR)。通過比對商業(yè)試劑盒Identifiler這4個基因座的分型結(jié)果、分析高度降解DNA的能力和抵抗PCR抑制物的作用，發(fā)現(xiàn)miniSTR分型結(jié)果與商業(yè)試劑盒Identifiler的結(jié)果相同；miniSTR比商業(yè)試劑盒更能從高度降解DNA中得到STR基因座分型結(jié)果，在PCR反應(yīng)時，能從更高濃度的抑制物(血紅蛋白)中得到分型結(jié)果。法醫(yī)可行性研究結(jié)果顯示，該復(fù)合擴(kuò)增體系的靈敏度達(dá)到200pg，分型結(jié)果穩(wěn)定，重復(fù)性好，能夠?qū)ΤＲ娀�質(zhì)上的斑痕正確分型，具有較好的組織同一性和種屬特異性。用于實(shí)際檢案，可以提高DNA高度降解檢材分型成功率。 構(gòu)建的5個SNP復(fù)合擴(kuò)增體系，片段長度在60-70bp之間。應(yīng)用多重引物單堿基延伸熒光檢測技術(shù)進(jìn)行分型。結(jié)果發(fā)現(xiàn)SNP比miniSTR從高度降解DNA中得到分型結(jié)果的成功率更高，并且能從含更高濃度抑制物(血紅蛋白)的樣本中得到分型。法醫(yī)可行性研究顯示，檢驗(yàn)的最低模板量為100pg，分型結(jié)果穩(wěn)定，重復(fù)性好，對法醫(yī)常見生物檢材，包括毛發(fā)、血痕、精斑、煙蒂等均獲得正確結(jié)果，并且可以判斷檢材來源者的性別。通過分別對20名無關(guān)男性個體和20名無關(guān)女性個體分型，得出5個SNP的等位基因頻率分布資料。所有常染色體SNP的雜合度均大于0．43，顯示出良好的多態(tài)性。 構(gòu)建的PCR內(nèi)對照物，所產(chǎn)生的片段大小為83bp，與商品化STR復(fù)合擴(kuò)增試劑盒內(nèi)標(biāo)為同種熒光染料標(biāo)記，能與商業(yè)試劑盒一同擴(kuò)增、電泳分離及熒光檢測，不影響商業(yè)試劑盒分型結(jié)果的準(zhǔn)確性、靈敏度和種屬特性等特征�？梢酝ㄟ^觀察內(nèi)對照產(chǎn)物峰高情況，，評估PCR反應(yīng)中抑制物的存在和作用情況。為指導(dǎo)DNA提取和PCR擴(kuò)增提供參考。 結(jié)論本課題構(gòu)建了一些有利于分析高度降解DNA的方法，彌補(bǔ)了商品化STR復(fù)合擴(kuò)增試劑盒在分析高度降解DNA的不足。為法醫(yī)高度降解檢材的DNA分析提供了新方法和新指標(biāo)。
[Abstract]:In order to improve the analysis of highly degraded DNA samples the success rate, to explore some new methods for analysis of highly degraded DNA, a new index is necessary. The purpose of this study is to construct some methods for analysis of highly degraded DNA, to compensate for the commercialization of STR multiplex kit in the lack of analysis of highly degraded DNA, increase the forensic the ability to identify.
From the practice of forensic cases in selected commercial STR multiplex amplification assay kit DNA the lowest success rate of 4 STR loci, binding sites by modifying primers, amplified fragment length decreases, the construction of a multiplex fluorescence detection and automatic classification system (miniSTR) type. MiniSTR and commercial STR composite amplification kit results are consistent, and the ratio of anti PCR inhibitor anti degradation ability of the experimental basis, DNA analysis technology working group (TWGDAM) guidelines for forensic feasibility study.
Select the 5 single nucleotide polymorphism loci (SNP) as the research object, 3 (rs999842, rs922992, rs924181) is located at chromosome, a (rs997262) located on chromosome X, a (M9) on chromosome Y. It constructed a short fragment length PCR multiplex amplification system, using multiple the single nucleotide extension fluorescence detection method for classification of anti degradation and anti PCR inhibitor ability comparison experiments with the miniSTR and forensic feasibility study on population genetics and sex determination.
Construction of a PCR control, so that it can work with commercial STR composite amplification kit together with amplification, electrophoresis, automation classification. Through the observation to the internal control product of peak height, to determine the presence and role of inhibitor in the PCR reaction. Combined with case of forensic feasibility assessment.
Results through the statistical 220 forensic biological evidence samples DNA typing results, found that the commercialization of STR composite amplification kit of D7S820, D18S51, CSF1P0, D2S1338, FGA five genotype success rate is the lowest. The core sequence repeat motif of the FGA is too long, it can not be amplified fragment length. To the ideal level, is not suitable for small fragments of STR design. Then we establish a D7S820 containing D18S51, CSF1P0 and D2S1338, a total of 4 loci multiplex fluorescence detection system (miniSTR). By comparing the 4 commercial Identifiler kit for genotyping results of analysis of highly degraded DNA ability and resistance to PCR inhibitors, found miniSTR results and Identifiler commercial kit for the same result; miniSTR can get from highly degraded DNA STR genotype were more than the commercial kit in the PCR reaction, the more The high concentration of inhibitor (HB) in genotyping results. Forensic feasibility study results showed that the sensitivity of multiplex PCR genotyping results reached 200pg, stability, good repeatability, capable of common matrix on the spot right type, with better organization identity and species specificity for the actual inspection. The case, DNA can be improved highly degraded classification success rate.
The construction of the 5 SNP multiplex amplification system, fragment length in the range of 60-70bp. Application of multiplex fluorescence detection of single base extension type. The results showed that SNP miniSTR from highly degraded DNA typing results obtained higher success rate, and from a higher concentration of inhibitor containing (hemoglobin) received the samples the feasibility study shows. Forensic testing for the minimum amount of template, 100PG, stable typing results, good repeatability, the common forensic biological samples, including hair, blood, semen, cigarette butts are getting the correct results, and can determine the source materials of the gender. By respectively in 20 unrelated male individuals and 20 unrelated female individual type, the distribution of allele frequency data of 5 SNP. The heterozygosity of all the autosomes of SNP was greater than 0.43, showed high polymorphism.
The construction of PCR control, the fragment size is 83bp, and commercial STR composite amplification kit with fluorescent dye labeled as internal standard, with commercial kit with amplification, electrophoresis and fluorescence detection, does not affect the accuracy of commercial kit genotyping results, sensitivity and species characteristics etc. features can be observed through the internal control product of peak height, assess the presence and role of inhibitor in the PCR reaction. In order to provide reference for DNA extraction and PCR amplification.
Conclusion this study has constructed some methods to analyze highly degraded DNA, and made up for the shortage of commercialized STR multiplex kit in the analysis of highly degraded DNA. It provided new methods and new indicators for DNA analysis of highly degraded forensic materials.

【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】：2007
【分類號】：D919

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 杜宏;張林;周斌;張海軍;梁偉波;沈月華;;微測序技術(shù)檢測12個Y-SNP及其遺傳多態(tài)性[J];法醫(yī)學(xué)雜志;2006年02期

本文編號：1695586