miniSTR复合扩增体系的研制与开发

发布时间：2018-06-27 11:45

本文选题：miniSTR + non-CODIS基因座　；参考：《河北医科大学》2009年博士论文

【摘要】： 目的:在法医DNA检验中,STR技术已经广泛用于个人识别和亲权鉴定方面。但是法医检案工作中,常常由于高温、潮湿、暴晒、微生物等因素的影响,使检材中的DNA分子被损坏,发生断裂,分子变小,大片段丢失。在这些情况下应用STR技术不能成功地得出结论,经常会出现“优势扩增”或者“无效扩增”,这就给正确的DNA分型带来困难。重新设计引物,减小扩增产物片段长度是目前解决DNA高度降解检材检验问题的一种新的方法。2001年9·11恐怖事件时这种方法被应用于遇难者的尸源认定,同时被正式命名为miniSTR技术。目前,AB公司已经开发出针对CODIS系统以内8个基因座的miniSTR商品化试剂盒。但由于CODIS系统内一些基因座侧翼序列不适合设计新的引物,或者等位基因范围太大,使这些基因座的扩增片段长度难以缩短到理想长度(125bp),因而并非CODIS系统内所有的STR基因座都适用于设计成miniSTR基因座。为解决这个问题,增加系统效能,非常有必要寻找CODIS系统之外的更多的miniSTR基因座,并进行人群调查。因此,本实验的目的是在数据库中筛选出适合中国汉族人群特征的miniSTR基因座,然后将其构建成新的miniSTR荧光标记复合扩增体系;并按照美国DNA分析方法技术工作组(TWGDAM)的指导方案,对复合扩增系统的灵敏度,基因座的种属特异性,案例应用,降解模型,陈旧样本等法医学应用进行初步研究。最终构建成符合中国汉族人群特征的miniSTR复合扩增体系,从而推动法医DNA试剂盒的国产化进程。方法:①参考Coble等的引物,在中国河北汉族120名无关个体中进行26个miniSTR基因座PCR扩增,获得河北汉族群体遗传学数据,根据数据初步筛选出8个多态性较高、片段较短的miniSTR基因座;在中国其他地区754个无关个体中进行这8个miniSTR基因座PCR扩增,扩增产物使用聚丙烯酞胺凝胶电泳分离,银染法显色,获得8个基因座的群体遗传学数据和各基因座的常见等位基因。②采用国产DNA聚合酶构建这8个miniSTR基因座的复合扩增体系,利用荧光复合扩增技术扩增,ABI310/3130基因分析仪对产物进行检测,Genemapper3.2软件分析结果;根据不同引物浓度、Mg~(2+)浓度、不同退火温度、不同循环次数下复合扩增的效果对复合体系进行优化。③根据群体遗传学研究的结果,应用分子克隆技术构建各基因座等位基因标准分型物,并根据国际法医遗传学会(international society of forensic genetics, ISFG )推荐的命名原则对各等位基因进行命名,同时根据检测数据进行统计分析编制出miniSTR复合扩增体系的基因分型模版。④对该荧光标记复合扩增体系的灵敏度、种属特异性、可重复性及对陈旧、腐败降解检材DNA的检测能力进行了研究;用实际案例进行验证,并与商品化试剂盒进行比较。结果:①成功构建了两个荧光标记miniSTR复合扩增体系,其中复合体系1包含D10S1248, D2S441, D1S1677和D9S2157四个基因座,复合体系2包含D9S1122, D10S1435, D12ATA63, D2S1776和Amelogenin五个基因座。应用该体系对300份中国汉族无关个体样本进行检验分析,经群体遗传学计算,该系统的累积个人识别率和非父排除率分别为0.99999999228和0.98547130732。②本实验构建的两个复合扩增体系条件易于优化,采用国产Taq酶进行扩增,即可得到满意的扩增产物和分型结果。相对于国外昂贵的试剂盒,它是一种成本低廉但效率高的miniSTR基因座复合扩增体系。③应用分子克隆技术,对9个基因座的64个等位基因进行克隆,经过调整各基因座等位基因的含量,构建出了两组复合体系的等位基因标准对照体系。根据实验数据和测序的命名结果设置基因分析命名参数指标,建立了两个荧光标记miniSTR复合扩增体系的基因分型命名模版,可以对样本准确分型。④法医应用性研究证明该体系具有良好的种属特异性,除恒河猴在Amelogenin出现色谱峰外,其余8个基因座在8种常见动物中均无特异性产物峰出现;对同一个体的不同组织检测结果一致;在0.125ngDNA模板量的情况下,可对9个基因座进行正确分型;通过对陈旧样本和降解模型的研究证明,该体系对降解检材有良好的扩增效果,较常规大片段STR试剂盒扩增成功率高,可用于常规试剂盒扩增失败的降解样本的分型;通过8个实际案例证明,该体系能用于法医学实践中,结果与鉴定结论一致。结论:本实验成功的构建了适合中国汉族人群的两个荧光标记miniSTR复合扩增体系。该荧光标记复合扩增体系可应用于ABI 310/3130检测平台,该体系扩增条件易于优化,成本低廉,分型结果稳定,种属特异性好,灵敏度和准确度高,对降解检材扩增成功率高,可以应用于法医学实际检案中,特别适合对陈旧检材和降解检材的DNA检测。为国内法医DNA分析提供了一个成本低,性能好的荧光标记miniSTR复合扩增检验系统,具有重大的应用价值。
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：D919

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