臺灣博碩士論文加值系統

本研究的主要目的為針對人類微量髮幹及指甲DNA的檢驗方法做評估研究。首先我們比較了phenol-chloroform及QIAamp DNA micro kit兩種萃取方法，不論髮幹或指甲樣本DNA以Real-time PCR的定量結果皆為phenol-chloroform的萃取方式所得DNA含量較高，且phenol-chloroform萃取出來的髮幹DNA其Real-time PCR定量數值在純化樣本體積稀釋5倍情形下不受黑色素的影響。反之，phenol-chloroform萃取的髮幹DNA在進行STR分型的PCR增幅反應時，卻需藉由稀釋3倍來減低PCR溶液中的黑色素含量，而得到較佳的分型結果。依文獻指出髮幹與指甲樣本皆有DNA裂解的現象，而我們發現在AmpFlSTR® Profiler Plus™的PCR增幅反應在28個循環數下分別需要10.53及1.32 ng DNA可做出完整STR分型，相當於需要髮幹重量4.68 mg、指甲重量0.02 mg，而將髮幹DNA進行STR的PCR增幅循環數增加到34，則髮幹DNA只需1.13 ng即可做出完整分型，相當於需要髮幹重量0.50 mg。髮幹近毛囊端所得DNA較髮幹末稍部分多，因此髮幹距離頭皮遠近亦會影響DNA萃取的多寡。髮幹樣本可進行粒線體HV1、HV2分析分別需要重量約1.11 x 10-3 mg及4.44 x 10-2 mg，而指甲樣本可進行粒線體HV1、HV2分析則分別需要重量約1.75 x 10-5及1.75 x 10-4 mg。這些研究成果將有助於未來使用指甲及髮幹做為檢體的刑事鑑定。

This main purpose of this study is to investigate the DNA analytical techniques for the specimens of human hair shafts and nails. We compared two kinds of DNA extraction methods, phenol-chloroform and QIAamp DNA micro kit. Either from hair shaft or from nail samples, the former method showed higher yield as quantitated by Real-time PCR. The inhibition of PCR reaction by melanin was not observed in the DNA quantitation by Real-time PCR in which the DNA sample solution from hair shafts was diluted 5-fold. However, for STR typing, the PCR reaction of AmpFIST® Profiler Plus was inhibited by melanin when the dilution of DNA sample solution from hair shafts was less than 3-fold. Previous study has found the degradation of DNA by nuclease in hair shafts and nails. This is further confirmed as the amounts of DNA extracted from hair shafts and nails required for STR typing using AmpFlSTR® Profiler Plus™ with 28 PCR cycles are 10.53 ng and 1.32 ng, respectively, corresponding to 4.68 mg hair shafts and 0.02 mg nails. When the PCR cycle number for the same PCR typing reaction was increased from 28 to 34, in contrast to 10.3 ng, only 1.32 ng DNA extracted from hair shafts was required to provide complete STR typing results. In addition, our study has suggested that the proximal part of hair shafts has a higher DNA content than the distal end has. For sequencing of mitochondrial HV1 and HV2 DNA, 1.11 x 10-3 mg and 4.44 x 10-2 mg of hair shafts and 1.75 x 10-5 and 1.75 x 10-4 mg of nails are needed, respectively. The results derived from this study will facilitate the forensic DNA analysis using samples of hair shafts and nails

目錄 I
表次 III
圖次 IV
圖次 IV
縮寫表 VI
摘要 VII
Abstract VIII
第一章 前言與目的 1
第一節 髮幹與指甲作為法醫鑑識證物的優缺點 1
第二節 phenol-chlorform & QIAamp DNA micro kit DNA萃取 2
第三節 Real-time PCR定量 3
第四節 STR 4
第五節 粒線體DNA定序 5
第六節 實驗目的與流程 5
第二章 材料與方法 7
第一節 實驗材料 7
一、髮幹與指甲樣品來源 7
二、試劑與緩衝液 7
三、酵素及樣品 8
四、主要儀器 9
第二節 實驗方法 10
一、髮幹、指甲DNA萃取 10
二、髮幹與指甲DNA 定量－Real-time PCR 13
三、STR分型 15
四、粒線體DNA定序 17
第三章 結果 21
第一節 樣本的蒐集 21
第二節 Phenol-chloroform萃取方法所得DNA含量較多 21
第三節 Real-time PCR定量數值並不受黑色素影響 23
第四節 不同根數髮幹稀釋1~5倍的分型結果 23
第五節 指甲、髮幹DNA進行完整STR分型的最小DNA含量 24
第六節 髮幹近毛囊端的DNA含量較高 26
第七節 可進行粒線體分析的最小染色體組(genomic)DNA含量 27
第四章 結論與討論 31
第一節 結論 31
第二節 Phenol-chloroform 萃取所得DNA含量較高 31
第三節 QIAamp DNA micro kit萃取所得DNA質較好 32
第四節 Real-time PCR不受黑色素影響 32
第五節 不同根數髮幹稀釋1~5倍的分型結果 33
第六節 指甲、髮幹DNA進行完整STR分型的最小DNA含量 33
第七節 髮幹近毛囊端的DNA含量較高 35
第八節 可進行粒線體分析的最小染色體組(genomic)DNA含量 35
第九節 未來研究方向 35
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