2015-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/695716摘要：在法醫基因體學領域中，去氧核醣核酸（DNA）短多重複序列（STR）標記多型性被常規性的應用作人身鑑別及血緣鑑定，但是，STR 標記往往需要較長的 DNA片段(200~500bp)，而法醫實務個案的 DNA檢體，常常是裂解的，所以這些常規性的方法有時得不到完整結果而無法判斷。近年來，單核苷酸多型性(SNP)被發現可應用於人身鑑別，應用 SNP 的分析，只需較短的 DNA片段(少於 150bp)即可判斷，所以，法醫案件中斷裂的 DNA片段也可以適用。發展新的 SNP 位置組合，用於人身鑑別及親緣鑑定是法醫學的新方向，我們已研究出一個具有高鑑別力包含 267個高變異性的 SNP 點位組合。在法醫實務工作，分析裂解且懸殊比率混合的 DNA檢體是重要議題，應用一般的DNA分析方法作混合 DNA 檢體之中的較微量者的 DNA型別非常困難，而次世代定序方法，可同時對數百萬個 DNA片段定序，因此可以分辨出混合的裂解 DNA 中較微量的 DNA成分，且作基因型判別。 我們研究的目的是以前期研究的 267 個 SNP 為基礎，自行研發組合新的約 500 個SNP 點位組合，用次世代定序分析出裂解 DNA 混合檢體中的較少量 DNA 的型別，作人別鑑定及血緣鑑定。在這 2 年計畫中，首先我們以前期研究為基礎，設計一個約 500個 SNP 的人別鑑定套組。第一年，我們將收集 30 位男性及 30位女性自願捐出者的 DNA檢體，其中約半數有近親關係，先作這 60個人的 STR 及 SNP 位置組合基因型分析，確認其親屬關係，再以定焦超音波粉碎儀裂解各 DNA檢體，再作以其中無親緣關係的 10位男性及 10位女性之檢體在女（男）：男（女）為 9：1、19：1、39：1、59：1、79：1及 99：1之比例下混合裂解的 DNA混合組共 60 組，再用次世代定序方法分析我們新建立的 500 個 SNP 點位組合，作較多量者及較微量者的基因型別分析，計算各 SNP 點位敏感度極限，我們將綜合不同 SNP 點位型別計算出人別鑑定能力，且分辨出效益較好的 SNP 點位，調整此 500 SNPs 套組的 SNP 點位。第二年，我們用已確認為近親的 10位男性及 10位女性之檢體在女（男）：男（女）為 9：1、39：1、59：1、79：1、 99：1 及 149：1 之比例下混合裂解的 DNA 混合組 60 組，再用次世代定序方法分析我們調整後的 500 個 SNP 點位組合，作較多量者及較微量者的基因型別分析，計算各 SNP 點位敏感度極限，也要綜合不同 SNP 點位型別計算出人別鑑定能力，還要應用這 500 個SNP 作混合檢體來源者的血緣鑑定，另外也要取得 10個真正腐屍的裂解 DNA混合檢體作這 500個 SNP 套組的次世代定序分析，以確認此方法在實務上可應用。 我們的研究結果將證實應用次世代定序方法及多重 SNP 點位套組，對比例懸殊混合的裂解 DNA 檢體中微量裂解 DNA 檢體的基因型別有判別能力，且對微量混合的裂解 DNA檢測、人身鑑別及血緣鑑定是有效的，此研究結果在法醫實務應用非常重要。<br> Abstract: Short tandem repeat (STR) polymorphic makers are routinely used for individual identification and parentage testing in forensic genomics. However, STR markers analyses are limited by the need for long amplification products (200-500bp), which limits the use of degraded samples in forensic casework. Single nucleotide polymorphisms (SNPs) have been promoted as useful genetic markers for human identification in recent years. Single nucleotide polymorphisms rely on short amplicons (i.e., less than 150bp), which allow for the use of degraded DNA samples in forensic casework and high-throughput genotyping technologies. Forensic genomic studies have described preliminary SNP panels for use in human identification and parentage testing. We have developed an efficient 267-SNPs markers panel with high discrimination power. In forensic casework, the genotyping of the minor component of a degraded DNA mixture sample is an important issue. It is very difficult to detect the genotype of the low ratio contributor using routine DNA analysis. The next generation sequencing (NGS) offers the possibility to sequence up to millions of DNA strands. Even in fragmented DNA mixture, alleles with the same length can be distinguished based on SNPs or different repeat structures. Therefore, the genotype of the minor component in a degraded DNA mixture can be identified using NGS. The aim of our study is to analyze the genotypes of the minor components of fragmented DNA mixtures using NGS and our newly developed individual identification panel with about 500 SNP loci, based on our previously established 267-SNPs panel. In this 2-year research, an individual identification panel with about 500 SNPs will be established based on our previous research. In the first year, we will totally collect DNA sample from 30 female and 30 male volunteer donors. Half of them are closely relativeness. The individuals STRs and SNPs genotypes will be analyzed. Ultrasonication was performed for DNA fragmentation. The DNA sample of 10 pairs of unrelated female (male) and male (female) will be mixed at 9:1, 19:1, 39:1, 59:1, 79:1 and 99:1 ratios. The 500 SNPs of these 60 samples of fragmented DNA mixture will be analyzed with NGS. The detection rate of the genotypes of the minor components at different ratio will be calculated. The resulted combined random match probability with be calculated. The 500 SNPs panel will be modified according to the effectiveness of each SNP. In the second year, the fragmented DNA samples of 10 pairs of closly related female (male) and male (female) will be mixed at 9:1, 39:1, 59:1, 79:1, 99:1 and 149:1 ratios. The modified 500 SNPs of these 60 samples of fragmented DNA mixture will be analyzed using NGS. The detection rate of the genotypes of the minor components at different ratio will be calculated. The resulted combined random match probability with be estimated. The relativeness of the donors of the DNA mixture will be analyzed based on this modified 500 SNPs panel. Another ten degraded DNA mixture sample from decomposed corpus of real forensic casework will by analyzed using NGS to validate the effectiveness of this 500 SNPs panel. Our findings will present the performance of genotyping and individual identification ability of both the major and minor DNA components in a fragmented DNA mixture using NGS and multiple SNP markers. This method will be proved to be effective in minor component DNA detection, individual identification, and relativeness identification (parentage testing) in fragmented DNA mixtures. It is very important in forensic casework.