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標題: | 高溫環境對於以牙齒內DNA做基因鑑定的影響 Effects of High Temperature Environment On Genetic Analysis Using DNA Isolated From Teeth |
作者: | Chen-Che Wu 吳政哲 |
指導教授: | 藍萬烘(Wan-Hong Lan) |
關鍵字: | 牙科法醫學,基因鑑定, forensic odontology,gene analysis, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 牙科法醫的鑑定方式通常是利用每個人不同的齒顎特徵,如同指紋的辨析一樣,可以利用牙科病歷內的治療記錄來做比對,如拔牙記錄,就會在牙弓中顯示出缺牙狀況;如根管治療,就會在X光片上顯示根管填充物;如贗復治療,就會在死者口中看到牙冠、牙橋、或活動假牙等物體。牙齒是人體中最硬的組織,並且被皮膚、肌肉、及上下頷骨所保護,可以對抗外力或環境對於死者的衝擊,所以牙齒可以在死者體內保存的很久,而且形狀仍可辨別。但是,如果遇到這些醫療記錄付之闕如,或是經過年久腐爛、屍塊殘缺不全、焚燒等情況,認屍的過程將會遭遇許多困難。所以在這種狀況下,抽取亡者身上組織的DNA,如血液、唾液、頭髮、口腔表皮細胞或者是牙齒,並利用DNA的多形性來做為法醫鑑識及比對,可以得到很好的效果。
隨機選取來牙科門診治療的病人,經病人的同意後,取得拔除的牙齒。第一部份實驗,我們利用QIAamp® DNA Micro Kit,測試是否能從牙周組織及牙髓組織中萃取出DNA,然後以PCR放大IL-β基因片段,接著在電泳膠上呈現。第二部分實驗,加入環境因子,模擬高溫加熱時,對牙齒內的DNA有何影響。我們把牙齒分成十組,以不同的溫度及時間加熱後,萃取出DNA,接著再以Real-Time PCR做定量。第三部分實驗,我們利用AmpFlSTR® Identifiler™ PCR Amplification Kit 檢視DNA內15個STR基因片段以及Amelogenin 性別鑑定基因的複製狀況,以瞭解高溫因子的破壞下,哪些長度的基因片段能夠保留下來。 綜合三個部分的實驗,本研究已經建立一套牙科法醫學的DNA檢驗流程。首先將收集來的牙齒樣本仔細清洗乾淨並做表面去污處理之後,再加以乾燥,並分別保存在室溫的環境下。萃取經過處理後牙齒的DNA時,應先將牙齒用高速磨粉機加以磨碎後,再以QIAamp® DNA Micro Kit萃取DNA。欲定量DNA,可以使用Real-Time PCR檢測DNA的濃度;欲定性DNA,可以利用STR system。本研究的環境因子為溫度,當溫度達到300℃,並加熱30分鐘以上,DNA的平均濃度僅剩1 到2 pg/μl,輔以STR system印證,確實已無任何鑑定價值可言。 Like fingerprint, the methods of forensic odontological identification usually utilize unique maxillary characteristics of each person from dental chart in clinic. In case of found bodies in which dental treatment is present, comparison of antemortem and postmortem dental records is a common appraisal technique. For example, if extraction was recorded, we will find the missing teeth in one’s arch. If pior endodontic therapy was performed, we will see the root canal filling material in x-ray film. Since dental prosthesis were noted, we will notice the bulk of crown, bridge, restoration , and so on in the oral cavity of a victim. Teeth are the hardest substances in the human body and protected by skin, muscle, and bone. Teeth will be completely conserved in a period of time because of resistance against crushing force or environmental factors. But it is hardly to identify when the dental charts were missing or the human remains were fragmented result from burning or crushing. In these circumstances, it is effective to apply DNA polymorphism technique to identify by extracting DNA from blood, saliva, hair, or teeth of human remains. The extracted teeth were obtained from department of dentistry in dental clinic. In first part of experiment, we used QIAamp® DNA Micro Kit for DNA extracting. Polymerase chain reaction was carried out by adding IL-1β primer and followed by gel electrophoresis to evaluate the amount and efficiency of the amplification. In second part of experiment, we tested the effects of high temperature environment on DNA analysis from teeth. We extracted DNA from teeth and quantified concentration of DNA by real-time PCR. In third part of experiment, we used AmpFlSTR® Identifiler™ PCR Amplification Kit to identify what STR fragments could be survive after the destruction of high temperature. In this research we set up a procedure of DNA analysis for forensic odontology. The teeth should be taken a series of washed, surface decontamination, dried, and stored in room temperature when collected. Preparing for DNA extraction by QIAamp® DNA Micro Kit, teeth should be grinded by machine. We could utilize real-time PCR for quantification of DNA concentration and qualify DNA by STR system. When temperature reached to 300℃ and be lasting 30 minutes, DNA concentration was less than 1 to 2 pg/μl. In this experimental condition, it was useless for DNA identification proved by STR system. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33976 |
全文授權: | 有償授權 |
顯示於系所單位: | 臨床牙醫學研究所 |
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