武漢漢族群體24個mtDNA編碼區(qū)SNPs研究
發(fā)布時間：2019-06-08 14:33
【摘要】：研究背景法醫(yī)案件的現(xiàn)場生物檢材,常因復(fù)雜環(huán)境因素的影響,核DNA容易發(fā)生降解,導(dǎo)致核DNA遺傳標(biāo)記檢測陰性。此外,犯罪現(xiàn)場遺留的常見檢材,例如:毛發(fā),牙齒,骨頭等,只含有少量DNA,分型檢測難度較大。所以,當(dāng)核基因組DNA的信息無法獲得時,檢測線粒體DNA(mtDNA)成為一常用的替代選擇。單個細(xì)胞中mtDNA拷貝數(shù)遠(yuǎn)比核DNA多,而且線粒體基因組堿基突變率約是核DNA的10倍。尤其位于非編碼區(qū),HV-I區(qū)和HV-II區(qū)含有密集的多態(tài)性位點(diǎn)。控制區(qū)中所存在的大量堿基序列差異使其成為進(jìn)行個人識別的主要方法。mtDNA具有母系遺傳和缺乏重組的特性,具有相對較高的隨機(jī)匹配概率。大樣本調(diào)查中發(fā)現(xiàn)大約84%的個體擁有唯一的HV-I和HV-II mtDNA序列,但仍有部分個體的序列相同。利用mtDNA多態(tài)性作個人識別的意義在于排除同一性。群體遺傳學(xué)研究發(fā)現(xiàn),mtDNA特征是可以按血緣關(guān)系分簇,或分成單倍群。單倍群是以分布于編碼區(qū)的特異性SNP位點(diǎn)來區(qū)分的,某些單倍群在特定人群中可呈分布廣泛。雖然高變區(qū)含有較多的單倍群,但在15,447 bp的編碼區(qū)也有相當(dāng)數(shù)量的多態(tài)性位點(diǎn)。由于編碼區(qū)承受的選擇壓力相對較大,因此這區(qū)段堿基序列的突變率更低,分析編碼區(qū)的多態(tài)性位點(diǎn)成為法醫(yī)線粒體DNA研究領(lǐng)域中,提高排除率的一種有用工具。 目的基于成熟的SNaPshot技術(shù)(微測序技術(shù))建立一套簡便、經(jīng)濟(jì)、高效的實(shí)驗(yàn)方案,調(diào)查武漢地區(qū)漢族人群mtDNA編碼區(qū)24個SNPs的頻率分布,為法醫(yī)學(xué)領(lǐng)域的相關(guān)應(yīng)用提供依據(jù)。 方法選擇多態(tài)性較好的24個mtDNA-SNPs,查明其堿基序列,使用相關(guān)軟件設(shè)計目的片段擴(kuò)增和單堿基延伸的兩套引物,首先利用等位基因特異性PCR技術(shù)擴(kuò)增不同基因座的等位基因片段,然后使用純化的PCR產(chǎn)物進(jìn)行SBE反應(yīng),最后通過熒光檢測識別所有基因座延伸的單個堿基類型,以此構(gòu)建24個mtDNA-SNP基因座的熒光復(fù)合擴(kuò)增檢測體系,并用建立的檢測體系對100名武漢漢族無關(guān)個體進(jìn)行檢測分型。 結(jié)果構(gòu)建了24個mtDNA-SNP基因座熒光復(fù)合擴(kuò)增體系,分型檢測結(jié)果表現(xiàn)為:各組SNP基因座具有顏色異同的八個產(chǎn)物峰,不同SNP基因座的片段大小不同,同一SNP基因座的不同堿基表現(xiàn)出峰型位置的差異。在檢測的100個樣本中,發(fā)現(xiàn)31個單倍型,其頻率范圍是0.01~0.14。24個mtDNA-SNP基因座在武漢漢族群體中的單倍型多樣性為0.952。 結(jié)論本研究所創(chuàng)建的mt-SNP復(fù)合擴(kuò)增體系具有簡便、經(jīng)濟(jì)、高效的特點(diǎn),具有較高的法醫(yī)學(xué)應(yīng)用價值。
[Abstract]:Background the field biological samples of forensic cases are often prone to degradation of nuclear DNA due to the influence of complex environmental factors, which leads to the negative detection of nuclear DNA genetic markers. In addition, the common materials left over from the crime scene, such as hair, teeth, bones, etc., contain only a small amount of DNA, typing. Therefore, when the information of nuclear genomic DNA is not available, the detection of mitochondrial DNA (mtDNA) has become a common alternative. The number of copies of mtDNA in a single cell is much more than that of nuclear DNA, and the base mutation rate of mtDNA is about 10 times higher than that of nuclear DNA. Especially in the non-coding region, HV-I region and HV-II region contain dense polymorphism sites. A large number of base sequence differences in the control region make it the main method for personal identification. MtDNA has the characteristics of maternal inheritance and lack of recombination, and has a relatively high random matching probability. In the large sample survey, about 84% of the individuals had unique HV-I and HV-II mtDNA sequences, but some of them still had the same sequences. The significance of using mtDNA polymorphism as personal identification is to exclude identity. Population genetics studies have found that mtDNA can be grouped according to consanguinity or divided into haploids. Haploids are distinguished by specific SNP loci distributed in the coding region, and some haploids can be widely distributed in specific populations. Although the hypervariable region contains more haploids, there are also a considerable number of polymorphism loci in the coding region of 15447 bp. Because the selection pressure of the coding region is relatively high, the mutation rate of the base sequence in this region is lower. The analysis of the polymorphism sites in the coding region has become a useful tool to improve the exclusion rate in the field of forensic mitochondrial DNA. Objective to establish a simple, economical and efficient experimental scheme based on mature SNaPshot technology (microsequencing) to investigate the frequency distribution of 24 SNPs in mtDNA coding region of Han population in Wuhan, so as to provide evidence for the application of forensic science. Methods 24 mtDNA-SNPs, with good polymorphism were selected to identify their base sequences, and two sets of primers were designed to amplify the target fragment and extend the single base by using the relevant software. First, the allelic fragments of different loci were amplified by allele-specific PCR, then the purified PCR products were used for SBE reaction, and finally the single base types extending from all loci were identified by fluorescence detection. The fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed, and the typing of 100 unrelated individuals in Wuhan Han nationality was detected by the established detection system. Results the fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed. the results of typing showed that there were eight product peaks with different colors in each group of SNP loci, and the fragments of different SNP loci were different. The peak position of different bases at the same SNP locus was different. Of the 100 samples tested, 31 haplotypes were found, the frequency range of which was 0.01 鈮，
本文編號：2495362
[Abstract]:Background the field biological samples of forensic cases are often prone to degradation of nuclear DNA due to the influence of complex environmental factors, which leads to the negative detection of nuclear DNA genetic markers. In addition, the common materials left over from the crime scene, such as hair, teeth, bones, etc., contain only a small amount of DNA, typing. Therefore, when the information of nuclear genomic DNA is not available, the detection of mitochondrial DNA (mtDNA) has become a common alternative. The number of copies of mtDNA in a single cell is much more than that of nuclear DNA, and the base mutation rate of mtDNA is about 10 times higher than that of nuclear DNA. Especially in the non-coding region, HV-I region and HV-II region contain dense polymorphism sites. A large number of base sequence differences in the control region make it the main method for personal identification. MtDNA has the characteristics of maternal inheritance and lack of recombination, and has a relatively high random matching probability. In the large sample survey, about 84% of the individuals had unique HV-I and HV-II mtDNA sequences, but some of them still had the same sequences. The significance of using mtDNA polymorphism as personal identification is to exclude identity. Population genetics studies have found that mtDNA can be grouped according to consanguinity or divided into haploids. Haploids are distinguished by specific SNP loci distributed in the coding region, and some haploids can be widely distributed in specific populations. Although the hypervariable region contains more haploids, there are also a considerable number of polymorphism loci in the coding region of 15447 bp. Because the selection pressure of the coding region is relatively high, the mutation rate of the base sequence in this region is lower. The analysis of the polymorphism sites in the coding region has become a useful tool to improve the exclusion rate in the field of forensic mitochondrial DNA. Objective to establish a simple, economical and efficient experimental scheme based on mature SNaPshot technology (microsequencing) to investigate the frequency distribution of 24 SNPs in mtDNA coding region of Han population in Wuhan, so as to provide evidence for the application of forensic science. Methods 24 mtDNA-SNPs, with good polymorphism were selected to identify their base sequences, and two sets of primers were designed to amplify the target fragment and extend the single base by using the relevant software. First, the allelic fragments of different loci were amplified by allele-specific PCR, then the purified PCR products were used for SBE reaction, and finally the single base types extending from all loci were identified by fluorescence detection. The fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed, and the typing of 100 unrelated individuals in Wuhan Han nationality was detected by the established detection system. Results the fluorescence compound amplification system of 24 mtDNA-SNP loci was constructed. the results of typing showed that there were eight product peaks with different colors in each group of SNP loci, and the fragments of different SNP loci were different. The peak position of different bases at the same SNP locus was different. Of the 100 samples tested, 31 haplotypes were found, the frequency range of which was 0.01 鈮，
本文編號：2495362
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