法医学降解检材DNA分析的新指标探索

发布时间：2018-04-01 12:25

本文选题：STR　切入点：miniSTR　出处：《四川大学》2007年博士论文

【摘要】： 目的为了提高分析DNA高度降解样本的成功率，探索一些针对高度降解DNA分析的新方法、新指标十分必要。本课题旨在构建一些有利于分析高度降解DNA的方法，以弥补商品化STR复合扩增试剂盒在分析高度降解DNA的不足，增加法医学个人识别能力。方法从法医检案的实践中挑选出商品化STR复合扩增试剂盒DNA分析成功率最低的4个STR基因座，通过修改引物结合区的位置，减小扩增片段的长度，将其构建一个复合扩增荧光检测和自动化分型体系(miniSTR)。将miniSTR与商品化STR复合扩增试剂盒的分型结果进行一致性、抗降解和抗PCR抑制物能力比对实验，依据DNA分析技术工作组(TWGDAM)指南进行法医学可行性研究。选择5个单核苷酸多态性基因座(SNP)作为研究对象，3个(rs999842、rs922992、rs924181)定位于常染色体，一个(rs997262)定位于X染色体，一个(m9)位于Y染色体。将其构建成一个超短片段长度PCR复合扩增体系，利用多重单碱基延伸荧光检测方法进行分型，与构建的miniSTR进行抗降解和抗PCR抑制剂能力对比实验，并进行法医学可行性、群体遗传学和性别判断的研究。构建一个PCR内对照物，使其能与商品化STR复合扩增试剂盒一同扩增，一同电泳，一同自动化分型。通过观察内对照产物峰高情况，确定PCR反应中抑制物的存在和作用情况。结合案例进行法医学可行性评估。结果通过统计220份法医生物物证样本的DNA分型结果，发现商品化STR复合扩增试剂盒对D7S820、D18S51、CSF1P0、D2S1338、FGA五个基因座分型的成功率最低。由于FGA的核心重复基序的序列太长，无法将其扩增片段长度减小到理想的程度，不适合进行小片段STR设计。于是我们建立了一个含有D7S820、D18S51、CSF1P0和D2S1338共4个基因座的复合扩增荧光检测体系(miniSTR)。通过比对商业试剂盒Identifiler这4个基因座的分型结果、分析高度降解DNA的能力和抵抗PCR抑制物的作用，发现miniSTR分型结果与商业试剂盒Identifiler的结果相同；miniSTR比商业试剂盒更能从高度降解DNA中得到STR基因座分型结果，在PCR反应时，能从更高浓度的抑制物(血红蛋白)中得到分型结果。法医可行性研究结果显示，该复合扩增体系的灵敏度达到200pg，分型结果稳定，重复性好，能够对常见基质上的斑痕正确分型，具有较好的组织同一性和种属特异性。用于实际检案，可以提高DNA高度降解检材分型成功率。构建的5个SNP复合扩增体系，片段长度在60-70bp之间。应用多重引物单碱基延伸荧光检测技术进行分型。结果发现SNP比miniSTR从高度降解DNA中得到分型结果的成功率更高，并且能从含更高浓度抑制物(血红蛋白)的样本中得到分型。法医可行性研究显示，检验的最低模板量为100pg，分型结果稳定，重复性好，对法医常见生物检材，包括毛发、血痕、精斑、烟蒂等均获得正确结果，并且可以判断检材来源者的性别。通过分别对20名无关男性个体和20名无关女性个体分型，得出5个SNP的等位基因频率分布资料。所有常染色体SNP的杂合度均大于0．43，显示出良好的多态性。构建的PCR内对照物，所产生的片段大小为83bp，与商品化STR复合扩增试剂盒内标为同种荧光染料标记，能与商业试剂盒一同扩增、电泳分离及荧光检测，不影响商业试剂盒分型结果的准确性、灵敏度和种属特性等特征。可以通过观察内对照产物峰高情况，，评估PCR反应中抑制物的存在和作用情况。为指导DNA提取和PCR扩增提供参考。结论本课题构建了一些有利于分析高度降解DNA的方法，弥补了商品化STR复合扩增试剂盒在分析高度降解DNA的不足。为法医高度降解检材的DNA分析提供了新方法和新指标。
[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.

【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919

【参考文献】