利用巨量平行定序方法分析裂解DNA之基因型別以進行人別鑑定及親緣鑑定

Abstract

法醫分子生物技術是人別鑑定與親緣鑑定主要方法，目前是以毛細管電泳為基礎的短相連重複序列 (STR) 分析為主，然而DNA因腐敗或被破壞而裂解時，用傳統毛細管電泳方法所做出的STR基因座分析，結果可能不足以作人別鑑定。近年因巨量平行定序方法 (MPS) 的發展，可以同時分析STR與單核苷酸多型性 (SNP)，除了可以得到法醫常規分析的STR基因座資訊之外，還可以將其重複序列進行定序以尋找其序列之變異性，對於腐敗或被破壞而裂解的DNA檢體分析是具有潛力的方法。本研究的目的在透過次氯酸鈉裂解DNA檢體或模擬法醫DNA檢體的分析，探討巨量平行定序方法分析裂解的DNA檢體作人別鑑定及親緣關係鑑定之正確性。 本研究收集來自21組家庭共50位台灣漢人的周邊血、臍帶血、病理切片、舊臍帶及牙齒檢體共81件，其中將部分的周邊血製作成血片並以次氯酸鈉浸泡破壞，而牙齒檢體則直接抽取DNA或是製成脫鈣的牙齒切片後再抽取DNA。分別進行傳統毛細管電泳方法與巨量平行定序方法分析，使裂解與未裂解參考檢體比對，進行人別鑑定及親緣鑑定計算。 研究結果顯示巨量平行定序分析27個體染色體STR、25個Y染色體STR、7個X染色體STR與96個體染色體SNP，其在未裂解參考檢體的分析上皆較毛細管電泳15個體染色體STR分析有更佳的鑑別力。經次氯酸鈉裂解的DNA檢體，雖然在巨量平行定序法上出現較多的對偶基因型改變，然而其在人別鑑定及親緣鑑定計算可以得到更低的隨機相符頻率及更高的累積似然比。而模擬法醫檢體，除了脫鈣的牙齒切片在本研究幾乎無法進行正確的分析之外，一般病理切片、臍帶、一般牙齒皆可得到巨量平行定序法較毛細管電泳法優異的統計結果。 此外本研究也完成了143個台灣地區漢人 (54個男性，89個女性) 以國際法醫遺傳學協會所建議之標準化命名方式進行命名的巨量平行定序 (MPS) 27個體染色體STR、25個Y染色體STR、7個X染色體STR及96個體染色體SNP之頻率資料庫，可以對我國未來的法醫DNA人別鑑定有所助益。 本研究的結論為巨量平行定序方法可以同時分析STR及SNP標記，分析裂解的DNA檢體，作人別鑑定與親緣鑑定，比毛細管電泳法只作STR標記有更好的鑑別效果。

Forensic molecular biotechnology is the main method for individual identification and relationship test. It is mainly analysis by short tandem repeat (STR) which is based on capillary electrophoresis. However, when DNA is degraded due to decompose or destroyed, the results of STR loci carried out by traditional capillary electrophoresis may be insufficient for individual identification. In recent years, due to the development of massive parallel sequencing (MPS), STR and single nucleotide polymorphism (SNP) can be analyzed at the same time. In addition to the information of STR loci that can be analyzed routinely, MPS can also sequence the repeated sequence to obtain the information of sequence variances. It is a potential method for the analysis of decomposed or destroyed DNA samples. The purpose of this study is to analyze the accuracy of the individual identification and relationship test by analyzing the DNA samples destroyed by sodium hypochlorite or mock forensic DNA samples. In this study, a total of 81 samples were collected from 50 Taiwanese Han people of 21 families. These samples included peripheral blood, umbilical cord blood, pathological sections, dried umbilical cord and dental specimens. Dried blood spots were made and soaked with sodium hypochlorite to destroy DNA. DNA of the dental specimens were extracted directly or extracted after decalcified tooth slices were prepared. The traditional capillary electrophoresis method and MPS method were performed to analyze the degraded and nondegraded samples, and the individual identification and relationship test were also performed. The results show that MPS method with 27 autosomal STRs, 25 Y-STRs, 7 X-STRs and 96 autosomal SNPs analysed has better discrimination in the analysis of nondegraded samples than using capillary electrophoresis method with 15 autosomal STRs analysed. Although the DNA samples destoryed by sodium hypochlorite showed more alleles changed in the MPS method, the individual identification and relationship test still showed better discrimination than capillary electrophoresis method. Except for decalcified tooth slices, which can hardly be accurately analyzed in this study, mock forensic DNA samples included pathological sections, dried umbilical cords, and teeth could obtain excellent statistical results from MPS method compared to capillary electrophoresis method. In addition, the allele frequency database for MPS of 143 random Taiwanese Han people (54 males, 89 females) was also described in this study. The alleles of this database included 27 autosomal STRs, 25 Y-STRs, 7 X-STRs and 96 autosomal SNPs, which were named after the standardized naming method recommended by International Society for Forensic Genetics. The database would be helpful to Taiwan in individual identification. In conclusion, MPS method is able to analyze the degraded DNA samples for individual identification and relationship test.