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研究生:蘇英圖
研究生(外文):Ying-Tu Su
論文名稱:微量乳化之應用
論文名稱(外文):Application of MicroEmusification:Emulsion Polymerase Chain Reaction
指導教授:沈志忠沈志忠引用關係
指導教授(外文):Jyh-Jong Sheen
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:機械與機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:乳化聚合?反應DNA模板暫態過程微量乳化
外文關鍵詞:ePCRDNA templatetransient processtiny-volume micro-emulsification
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本研究是利用軟微影技術,使用聚二甲基矽氧烷(PDMS)製作微量乳化晶片,並將其應用於乳化聚合?反應(emulsion Polymerase chain reaction, ePCR)技術上。乳化聚合?反應是將DNA溶液,經過乳化後以液滴的形態均勻散佈於油相中,並以液滴作為PCR的反應空間。如此,可使原本反應空間裡存在多種類型DNA模板,藉由乳化作用提高其DNA模板類型的一致性,故可降低競爭性序列干擾,並提升其放大效果。
有別於一般乳化晶片,本研究將針對ePCR所衍生之技術問題作出適當之處理與改善,以避免微量試劑因暫態過程造成乳化液滴過大或大小不一。本研究的做法為:(1)先以一水溶液注入,產生液滴避免DNA試劑經歷液滴產生的暫態過程;(2)利用水溶液與氣泡前後包夾微量的DNA試劑,再進行DNA試劑的乳化;(3)搭載薄膜氣動閥門進行水溶液與DNA的控制。
微量乳化晶片之特性實驗,包括液滴之均勻度、尺寸以及耐熱度進行前置性實驗。此晶片所製備之液滴尺寸可達到10 μm以下,且液滴之差異變異係數最小約為2%。最後進行ePCR實驗,由單一模板進行PCR的電泳圖,可看出複製片段之精度顯然較一般PCR為準確。多模板之ePCR電泳圖亦得到相似的結果。
關鍵字:乳化聚合?反應、DNA模板、暫態過程、微量乳化
In this thesis, soft lithography technique was used to produce a micro emulsion chip. This chip can handle tiny volume of DNA solution for polymerase chain reation (ePCR). The DNA solution is dispersed in the emulsified droplets in a continuous oil phase, and the droplets become reaction space for PCR. As a result, the consistency of the multiple types of DNA templates existing in the original reaction space can be improved by emulsification process. Also, it will reduce competitive sequence interference and improve DNA magnification.
Differing from usual micro emulsification chips, this study focuses on the technical problems involved in dealing with tiny and expensive DNA solution in order to avoid non-uniform droplets in the transient of droplet generation. The main ideas are as follows: (1) Let water first go through transient process and produce water droplets; (2) Use water and bubble to place DNA solution in between, and pass it through emulsification hydrodynamic focusing channel; (3) Use pneumatic membrane valves control the stop and flow of water and DNAsolution.
Characterization of the emulsification chips includes droplet sizes, uniformity of droplets and hot-resistance of water-in-oil droplets. The smallest droplet diameter is less than 10 μm and the coefficient of variation is about 2%. The resulting electrophoretograms of both single or multiple templates’ PCR demonstrate that ePCR can improve the precision of amplified DNA segments. It is suggested from the experiment results that ePCR was proven to be efficient and could be used in further genetic research
Key word: ePCR, DNA template, transient process, tiny-volume micro-emulsification
中文摘要 i
Abstract ii
圖目錄 v
表目錄 vii
第一章 緒論 1
1.1 研究動機與目的 1
1.2 文獻回顧 4
1.2.1 微乳化晶片 4
1.2.2 乳化聚合?反應 7
1.3 文章架構 11
第二章 ePCR原理介紹 12
2.1 流體的黏滯性 12
2.1.1 表面張力 15
2.1.2 液滴之物理性質 16
2.2 乳化液的結構 17
2.3 微乳化液的穩定性 18
2.4 ePCR原理 21
第三章 微量乳化晶片之設計與製作 24
3.1 微量乳化晶片設計概念 24
3.2 晶片製作 29
3.2.1 主流道製作 29
3.2.2 空氣流道與晶片整體製作 30
第四章 實驗結果與討論 32
4.1 實驗設備與材料 32
4.1.1 實驗操作步驟 33
4.2 ePCR晶片乳化實驗結果 36
4.3 ePCR結果與討論 39
4.3.1 單一模板PCR之結果 40
4.3.2 多重模板PCR之結果 41
4.3.3 點帶石斑魚基因組進行ePCR之結果 42
第五章 結論與未來展望 44
5.1 結論 44
5.2 未來展望 45
參考文獻 46
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