摘要

本研究說明藉由PCR複製細胞核SSU rDNA之18S rDNA保守區域（使用NS1-NS2引子對）及ITS1、ITS2等可變區域（ITS1使用primer 2/primer 5引子對、ITS2使用primer 3/primer 4引子對）之DNA片段，並以3﹪agarose gel electrophoresis分析為鑑定方法，作為鑑識人體組織樣本是否含有海洋藻類（綠球藻Chlorella sp. 及擬球藻Nannochloropsis oculata）之可能性。結果以NS1-NS2 引子複製之PCR產物分別為綠球藻550 bp、擬球藻550 bp，人類600 bp；以ITS1複製之PCR產物分別為綠球藻有一個片段（350 bp），擬球藻有二個片段（400 bp及450 bp），人類有四個片段（350 bp、450 bp、500 bp及580 bp）。以ITS2複製之PCR產物分別為綠球藻有一個片段（430 bp），擬球藻有二個片段（430 bp及600 bp），人類有一個片段（400 bp）。使用人類特定引子組（AmpFlSTR® Profilerä PCR Amplification Kit）PCR結果，只有人類DNA可被認知，因此在含有綠球藻及擬球藻之DNA人類DNA樣本中，綠球藻及擬球藻是無法偵測到。所以在使用人類特定引子AmpFlSTR® Profilerä PCR Amplification Kit 的鑑驗方法是可以避免藻類生物跡證之干擾。擬球藻DNA之偵測極限為 10 pg，綠球藻為50 pg。結果顯示，以PCR 複製NS1-NS2片段可區分人類與藻類，但無法區分綠球藻與擬球藻；但以PCR 複製ITS1或ITS2片段則可區分綠球藻、擬球藻與人類。因此，利用可區分藻類及人類之特定DNA片段的PCR方法應可作為人在海洋環境溺死案件之死因調查的基礎。

Abstract

This study is to elucidate the possibility of DNA fragment identification method in the forensic detection of marine microalgae (Chlorella sp. and Nannochloropsis oculata) from human (Homo sapiens) body by 3% agarose gel electrophoresis of amplification fragments via PCR-amplified ribosomal gene small subunit (SSU) rDNA molecules as primers, which are specific nucleotide segments on conserved regions (18S rDNA region (NS1-NS2 primers))on SSU rDNA and on variable regions (internal transcribed spacer (ITS) regions (primer 2/primer 5 for ITS1 region and primer 3/primer 4 for ITS2 region)). NS1-NS2-amplified PCR fragments are 550 bp for C. sp., 550 bp for N. oculata and 600 bp for human. ITS1-amplified PCR fragments are 1, 2 and 4 bands for C. sp. (350 bp), N. oculata (400 and 450 bp) and human (300, 450, 500 and 580 bp), respectively, while ITS2-amplified PCR fragments are 1, 2 and 1 bands for C. sp. (430 bp), N. oculata (430 and 600 bp) and human (400 bp), respectively. By using human-specific primers (AmpFlSTR® Profilerä PCR Amplification Kit), only human can be identified in the sample containing C. sp., N. oculata and human DNA, whereas C. sp. and N. oculata can not be detected, indicating the prevention of algal interference in human-specific primer-PCR procedures via AmpFlSTR® Profilerä PCR Amplification Kit. Detection limits of C. sp. and N. oculata DNA were 50 and 10 pg, respectively. The results of present investigation show that algae can be distinguished from human by NS1-NS2-amplified PCR fragments but not between C. sp. and N. oculata, while C. sp., N. oculata and human can be distinguished by ITS1- or ITS2-amplified PCR fragments. Evidently, the specificity of DNA segments in marine microalgal and human DNA provides the base for investigation of cause of death in drowning case in the marine environment.

目 錄
第一章 緒論………………………………………………………………..….1
第一節 前言………………………….…….……………………………...…1
第二節 文獻回顧……………………………………………………..……...2
第三節 研究動機與目的…………………………….…………….……..…15
第二章 材料與方法………………………………….……………………….19
第一節 樣本取得………………………………………………….….……..19
第二節 實驗流程…………………….…………………………....……...19
第三節 DNA之萃取…………..…………………………………..………...19
第四節 定量分析………………….…………….………………..…….……24
第五節 PCR複製與電泳分析………….…………………....……………….25
第三章 結果與討論……………….……………………………………………32
第一節 PCR複製與電泳分析…………………..…………………..….…..32
第二節 DNA模板之偵測極限…………………………………………....….43
第三節 刑事鑑識應用及微藻跡證之採證要領與分子鑑識處理原則…....44
第四章 結論…………………………………………………….………………47
參考文獻…………..………………………………………………...…………49
附錄……………………………………………………………………………….57

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