泡水肋骨之DNA裂解情形之研究

硬骨組織由於結構組成特性，對於組織中DNA具有一定程度保護性；然在法醫實務上需待鑑定骨骸檢體常是於各種環境中尋獲，其品質狀況隨存在環境不同而有極大變異，使組織中DNA完整性亦受到不等程度損害，進而影響DNA分析成效；依據過往文獻研究，環境中水份、溫度與時間可能是左右硬骨組織DNA裂解程度的重要因素；然而對於浸泡在海水或淡水環境中的骨骸檢體，其影響硬骨組織DNA裂解的情形仍尚無相關研究系統性地分析比較。因此，本研究目的為，藉由將豬肋骨檢體與人肋骨檢體浸泡於海水與淡水環境中，並加入時間與溫度等變項，探討硬骨組織受到多種環境因素影響後，DNA裂解之情形與基因鑑定之可行性與信賴度。實驗分為豬肋骨與人肋骨兩部分進行，檢體共同經前處理後分別浸泡於4℃與室溫下的海水與淡水中；在不同時間階段結束浸泡後取出檢體粉碎，萃取DNA進行相關分析。豬肋骨實驗與人肋骨實驗皆以同步定量聚合酶連鎖反應 (qPCR) 進行DNA定量，並依據定量結果進行短相連重複序列基因分析 (STR)；其中人肋骨實驗若STR分析結果至少有八組以上基因座定型失敗，則增加10倍DNA模板量重新分析或進一步分析粒線體DNA D-loop區之hypervariable region 1 (mtDNA HV-1)。實驗結果顯示，肋骨檢體處於淡水環境或海水環境中時，組織核酸量隨浸泡時間增長而減低，且STR基因型別檢出情形亦隨不佳；然而，在含有緻密骨層較多之肋骨檢體與4℃低溫環境下，硬骨組織DNA裂解情形則較為減緩，顯示除環境溫度外，硬骨組織中所含有緻密骨層之多寡亦是重要影響因子。人肋骨檢體經過8個月的浸泡後雖仍可進行STR基因分析，但僅能得到部分基因圖譜且長度300 bp以上之基因座檢出皆不佳。對於可能為一高裂解狀態的DNA樣品，可增加PCR反應所需DNA量以提高STR基因座檢出情形，或藉由粒線體DNA基因分析，提供輔助鑑定。

關鍵字

DNA分析；肋骨；淡水；海水； STR ；粒線體DNA D-loop HV-1

並列摘要

With the characteristics of hard structure and compositions, bone provides a good protection of DNA in bony tissue. In forensic cases, skeletal remains of victims are frequently recovered in various environments. Normally the DNA from remains is severely degraded, thereby affecting the effectiveness of DNA analysis. Based on previous literature, the influentical factors for DNA degradation in bony tissue are humidity, temperature or timing. However, there are no systematic studies on the observation of DNA degradation about bony tissue immersed in freshwater or seawater. The aim of this study was to investigate the degradation of DNA and the reliability of DNA identification on pig ribs and human ribs immersed in seawater or freshwater for different time periods and temperature. Bone specimens were prepared and submerged in freshwater and seawater respectively, then kept in both 4℃ and room-temperature (25℃). After different time periods of immersion, DNA was extracted by crushed procedure, followed by qPCR quantification and STR typing studied. If more than 8 STR loci were failed to amplify in the study of human ribs, 10-fold DNA template was used to re-analysis and mtDNA HV-1 DNA was sequenced. The results showed that the amounts of DNA extracted from both kinds of ribs immersed in freshwater or seawater were decreased with time periods. The STR typing was also worsend. However, for those with more compact bone such as pig ribs or both pig ribs and human ribs kept in 4℃, the trend of DNA degradation would be retaded. This indicated that the density of compact bone and environmental temperature were important factors in the DNA degradation of bony tissue. STR profiles could be obtained in human ribs immersed even after 8 months. It showed, however, only partial STR profiles were obtained and the STR loci with 300 bp in length were all poorly detected. For the highly degraded DNA samples, the detection of STR loci could be improved by raising the amount of DNA template in PCR reaction, or sequencing the mtDNA D-loop to assist victim identification.

並列關鍵字

forensic DNA analysis ； rib bone ； water ； seawater ； STR ； mtDNA D-loop HV-1

參考文獻

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泡水肋骨之DNA裂解情形之研究

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