本論文首先介紹PCR-變性梯度膠電泳(polymerase chain reaction-denaturing gradient gel electrophoresis，PCR-DGGE)的原理及沿革，其次詳細描述PCR-DGGE 在石油污染整治過程監測的運用、在毒物污染整治過程監測上的運用、以及在其他領域進行的微生物菌相分析，最後並對其未來發展提出一綜合性的評論。PCR-DGGE 技術最初被使用來研究DNA 突變。由於它是一個非常有力的工具，能夠偵測在被PCR 放大的DNA 片段上的微小差異，因此經過正確的設計，對DNA 序列上的變化偵測效果能夠達到百分之百。DGGE 的分離原理是根據DNA 序列的不同，使DNA 樣品在變性膠體中產生不同程度的變性，而影響其在變性梯度膠體中的移動速度，而加以分離。PCR-DGGE 技術能有效偵測環境中微生物種類的分佈，包括還不能被培養的微生物菌種，因此已成為研究微生物菌相結構上應用最廣的分子技術之一。微生物學家們能利用此技術更深入了解環境中的微生物生態分佈，以及污染整治過程中微生物菌相的變化，如石油污染及其他毒物污染的整治等。此外，PCR-DGGE 也能應用在食品工業、農業及生物腸道等等的微生物生態研究上。當我們對於菌相分佈及優勢菌種有非常深度的瞭解後，便能對特定菌種進行分離、純化與培養，並且能夠充分利用這些菌種。關於PCR-DGGE 技術未來的發展，主要可分為三個方向，分別是前置作業的改善、DGGE 相關工具的研發以及與不同技術的整合運用。改善前置作業能使DGGE 對菌相的分析更加完善及準確，並且能增加DGGE對DNA 的敏感度及幫助其膠體的正常化等等。研發DGGE 相關工具可以提升其監測菌相的能力，並且能幫助我們更了解DGGE 電泳圖所代表的意義。而透過DGGE 與不同技術的整合運用，能夠使我們對微生物菌相的分佈、變動及功能等等有更深層的了解。

This thesis introduced the principle and the evolution of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), described its usage in monitoring the remediation process for oil and other chemical pollution as well as analyzing the nature microbial community, and finally made a comprehensive comment to its future development. PCR- DGGE was initially developed to study DNA mutations because it is a powerful tool to detect DNA base mutations in a PCR-amplified DNA fragment. Its accuracy can be as high as 100%. The principal of DGGE is based on the differences in DNA sequence that affect the melting points of each amplicons, and caused a decrease in the electrophoretic mobility of a partially melted DNA molecule in a polyacrylamide gel containing a linearly increasing gradient of DNA denaturants. PCR-DGGE can effectively detect the community structure of microorganisms in the environments, including the unculturable microorganisms. Nowadays, it has become one of the most frequently applied techniques to study the community structure of microorganisms. Microbiologists can use this technique to understand the microbial ecology, the shift of the microbial community structure during a bioremediation process, such as the oil pollution and other toxic chemical pollution. In addition, PCR-DGGE can also be used for the fermentation studies in food industry and agricultural industries. We can even identify unculturable microorganism by analyzing the DNA fragment sequences. This can help us to design a suitable medium to culture and isolate these ‘unculturable organisms’. There are three directions for the further development of PCR-DGGE technique: 1.Improvement of material preparation processes; 2. development of new DGGE-related tools; and 3. combination of other technology with DGGE. The improvement of preparation processes can make DGGE more accuracy to analyze the community structure of microorganisms, improve the sensitivity of DGGE fingerprint detection, and help the DGGE normalization. The development of DGGE-related tools can overcome the limitations imposed on DGGE by co-migration and help us to have a better understanding of the meanings for a DGGE pattern. The combination of other technology with DGGE can help us to have a clear understanding of the microbial ecology, the shift of the microbial community structure, and the function of microorganisms.

中文摘要……………………………………Ⅰ
英文摘要……………………………………Ⅲ
第一章 DGGE的原理與沿革……………………………1
1.DGGE的原理………………………….1
2.DGGE的沿革………………………………………3
2.1 鑑定基因的突變………………………………3
2.2 鑑定特定細胞及研究基因的多形性………………5
2.3 微生物菌相的觀察研究……………………………6
3. 實施DGGE電泳的條件………………………………11
第二章 DGGE 在環境微生物菌相分析的應用方法……13
1.土壤微生物genomic DNA萃取………………………13
2.DNA 萃取物的純化…………………………………15
3.PCR for DGGE………………………………………15
4.Gel elution………………………………………18
5.DGGE…………………………………………………19
5.1 製膠………………………………………………19
5.2 電泳………………………………………………19
6.螢光染色……………………………………………19
7. DGGE 的Fingerprinting pattern 統計分析…20
8.DNA定序……………………………………………21
9. 繪製親緣演化樹…………………………………21
第三章 石油污染的介紹及整治過程中DGGE 在監測上的運用………………………………………………………26
1.石油污染物的介紹…………………………………26
1.1 關於BTEX…………………………………………27
1.2 關於MTBE…………………………………………29
1.3 關於TMB …………………………………………30
1.4 關於TPH………………………………………… 30
1.5 石油降解的因子…………………………………33
2. 應用DGGE 監測石油污染整治的過程……………33
2.1 使用DGGE監測受石油污染的海洋環境的整治工作…………………34
2.2 使用DGGE觀察真菌對石油污染整治的影響……36
第四章 DGGE在毒物污染整治過程監測上的運用……38
1. 除草劑的污染………………………………………38
2. 掩埋場的滲漏液污染………………………………39
3. 氨的污染……………………………………………41
4. 氯化物的污染………………………………………43
5. 重金屬的污染………………………………………46
第五章 DGGE在其他領域進行的微生物菌相分析……48
1. 在農業上的運用……………………………………48
1.1 不同處理方式對微生物菌相的影響…………… 48
1.2 使用不同肥料所造成的影響…………………… 50
2. 環境生態上的研究…………………………………54
2.1 出海口及河流的生態研究……………………… 54
2.2 陸地生態研究…………………………………… 57
3. 腸道生態……………………………………………61
4. 食品的研究…………………………………………63
第六章 DGGE方法之改進與未來發展…………………64
1. 改進其前置作業……………………………………65
1.1 DNA萃取方法……………………………………………………… 65
1.2 PCR 引子的設計………………………………… 68
2. 研發DGGE 相關工具……………………………… 73
2.1 DGGEGE…………………………………………… 73
2.2 分析方式………………………………………… 74
3. 與不同技術的整合運用……………………………77
3.1 與TTGE 及CDGE 的整合運用…………………… 77
3.2與培養基方法的整合運用…………………………79
3.3 與蛋白質分析技術的整合運用………………… 80
4. 結論…………………………………………………82

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