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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳炳煇(Ping-Hei Chen) | |
dc.contributor.author | Tzu-Cheng Yang | en |
dc.contributor.author | 楊子正 | zh_TW |
dc.date.accessioned | 2021-06-16T10:22:50Z | - |
dc.date.available | 2013-08-20 | |
dc.date.copyright | 2013-08-20 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-16 | |
dc.identifier.citation | [1] Chou, W. P., Chen, P. H., Miao, M., Kuo, L. S., Yeh, S. H., and Chen, P. J., 'Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater,' Biotechniques, vol. 50, pp. 52-57, 2011.
[2] Krishnan, M., Ugaz, V. M., and Burns, M. A., 'PCR in a Rayleigh-Benard convection cell,' Science, vol. 298, pp. 793-793, 2002. [3] 張維真,兩種新型毛細管熱對流聚合酶連鎖反應機台的開發與研究,碩士 ,機械工程學研究所,臺灣大學,台北市,2011. [4] Wittwer, C. T, Herrmann, M. G., Moss, A. A., and Rasmussen, R. P., 'Continuous fluorescence monitoring of rapid cycle DNA amplification,' Biotechniques, vol. 22, pp. 130-1, 134-8, 1997. [5] Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T., 'Product differentiation by analysis of DNA melting curves during the polymerase chain reaction,' Anal Biochem, vol. 245, pp. 154-60, 1997. [6] Mao, H., Holden, M. A., You, M., and Cremer, P. S., 'Reusable platforms for high-throughput on-chip temperature gradient assays,' Anal Chem, vol. 74, pp. 5071-5, 2002. [7] 蘇筱涵,新型核酸解鏈曲線之技術開發與應用,碩士 ,機械工程研究所,臺灣大學,台北市,2009. [8] Marmur, J. and Doty, P., 'Determination of Base Composition of Deoxyribonucleic Acid from Its Thermal Denaturation Temperature.' Journal of Molecular Biology, vol. 5, pp. 109-&. 1962. [9] Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K., 'Hybridization of Synthetic Oligodeoxyribonucleotides to Phi-Chi-174 DNA - Effect of Single Base Pair Mismatch,' Nucleic Acids Research, vol. 6, pp. 3543-3557, 1979. [10] Nakano, S., Fujimoto, M., Hara, H., Sugimoto, N., 'Nucleic acid duplex stability : influence of base composition on cation effects' Nucleic Acids Research, vol. 27 pp. 2957-2965, 1999 [11] Howley, P. M., Israel, M. A., Law, M. F., and Martin, M. A., 'A Rapid Method for Detecting and Mapping Homology between Heterologous Dnas - Evaluation of Polyomavirus Genomes,' Journal of Biological Chemistry, vol. 254, pp. 4876-4883, 1979. [12] Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A., 'Predicting DNA Duplex Stability from the Base Sequence,' Proceedings of the National Academy of Sciences of the United States of America, vol. 83, pp. 3746-3750, 1986. [13] Borer, P. N., Dengler, B., Tinoco, I., and Uhlenbec.Oc,. 'Stability of Ribouncleic-Acid Double-Strand Helices,' Journal of Molecular Biology, vol. 86, pp. 843-853, 1974. [14] http://www.zgenebio.com.tw/gzr1gaq1z0.html# [15] Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N., 'Enzymatic Amplification of Beta-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle-Cell Anemia,' Science, vol. 230, pp. 1350-1354, 1985. [16] http://users.ugent.be/~avierstr/principles/pcr.html [17] Chandrasekhar, S., 'Hydrodynamic and Hydromagnetic Stability,' Clarendon, Oxford, 1961. [18] Rutledge, R. G., 'Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications.' Nucleic Acids Research, vol. 32, pp., 2004. [19] http://www.metrum.co.uk/thermography-r300-thermal-camera.htm [20] Roche Applied Science: June 2005, LightCycler 2.0 instrument operator's manual, software version 4.05. Chapter B:36 [21] http://en.wikipedia.org/wiki/SYBR_Green_I | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60596 | - |
dc.description.abstract | 解鏈曲線分析(Melting Curve Analysis , MCA)是利用DNA的雙股解鏈現象與螢光染劑亮度之關係,吾人可透過螢光訊號隨溫度變化得知該DNA之解鏈溫度,由於不同DNA之解鏈溫度不同,因此MCA可用來檢測檢體專一性。
市面上販售的商用MCA machine主要是以溫控系統對DNA檢體進行PCR擴增,擴增過程結束後再對DNA檢體進行MCA,但由於此種升溫系統十分精密,因此這些機台普遍有造價昂貴的缺點。 2011年Chou團隊開發出毛細管熱對流PCR[1] (Capillary Convection PCR , CCPCR),這種以單一溫控即可完成核酸擴增的方法,改善了傳統PCR機台溫控系統所需的昂貴成本,但CCPCR目前仍缺與MCA整合的系統。 本研究嘗試將CCPCR與空間式的MCA結合起來,將DNA檢體進行CCPCR後直接進行空間式MCA,在兩個過程中都是使用同一容器,以達到像Roche LightCycler2.0機台擁有核酸擴增與基因型分析的整合功能。 實驗先建立螢光偵測與加熱平台,然後決定毛細管內的溫度梯度,為了穩定毛細管內溫度場,因此本實驗使用紅銅鰭片來穩定溫度梯度。 最後針對三種DNA檢體進行MCA,將進行CCPCR後的試劑放置在螢光偵測平台,將拍出的螢光梯度對距離做一階微分取峰值,再對應到溫度梯度以決定檢體的Tm值,然後將結果與Roche LightCycler 2.0 和預測值比較,比較的結果顯示本研究與商用機台之結果之趨勢相符。 | zh_TW |
dc.description.abstract | The principle of Melting Curve Analysis (MCA) is to determine the specificity of amplicons through the relationship between intensity of fluorescence dye and the temperature of reagent in which the amplicons are produced. Commercial MCA machines are typically combined with a Polymerase Chain Reaction (PCR) process. After DNA templates have been amplified by PCR to the enough amount which can be detected, then the MCA process is turned on to determine the specificity of amplicons. Since a commercial MCA machine has very high accuracy of temperature control, the cost of machine is expensive.
Fortunately, Chen’s research group published and developed a Capillary Convection PCR (CCPCR) process to produce amplicons in 2011 [1]. This technique requires a single temperature control and can reduce the cost of PCR machine dramatically. However, their CCPCR machine is not equipped with MCA capability. In the past few years, some researchers used spatial temperature gradient to perform MCA, the advantage of which is the low cost of temperature control system and the short detection time. This thesis attempts to combine CCPCR with spatial- temperature- gradient MCA solution. DNA templates are amplified by a CCPCR platform in which the specificity of amplicons can be determined by the MCA approach. Then, such a CCPCR platform can have the same capability as those of Roche LightCycler, a commercial PCR machine integrate with MCA solution. This thesis first established a melting curve detection platform, including heating surface and optical system. The temperature gradient of reagent in the capillary tube can be established by heating the bottom surface of capillary tube and by cooling the capillary through the surrounding air. For the stability of temperature gradient, this work introduces a trapezoidal copper fin to cover the capillary tube to obtain stable temperature gradient along the tube axis. Lastly, three different DNA samples were tested. The measured melting temperatures of three tested samples are compared with those obtained by the commercial machine Roche LightCycler 2.0, and predicted values by well-known formula. The comparison shows a good agreement between the present measured results and those by the commercial machine. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:22:50Z (GMT). No. of bitstreams: 1 ntu-102-R00522116-1.pdf: 2302105 bytes, checksum: a132d2885b03dc8254cbe5b1dd4f762e (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRCT iii 符號說明 v 目錄 vi 圖目錄 viii 表目錄 x 第一章 緒論 1 1.1 前言 1 1.2 文獻回顧 3 1.3 論文架構 6 第二章 研究原理與方法 15 2.1 解鏈曲線分析 (Melting Curve Analysis) 15 2.1.1 基本原理 15 2.1.2 分析方法 16 2.1.3 解鏈溫度的預測值 17 2.1.4 MCA與傳統基因型分析方法之比較 18 2.2 聚合酶鏈鎖反應 ( Polymerase Chain Reaction ) 19 2.2.1 PCR基本原理 19 2.2.2 影響PCR的因素 20 2.3 毛細管熱對流聚合酶鏈鎖反應 21 2.4 鰭片應用於溫度穩定性 21 2.5 實驗數據分析 22 2.5.1 影像擷取 22 2.5.2 曲線擬合方法 22 第三章 實驗設備與研究步驟 29 3.1 實驗設備 29 3.1.1 螢光量測系統 29 3.1.2 加熱系統 29 3.1.3 試劑容器 29 3.1.4 溫度量測系統 30 3.1.5 LightCycler 2.0 檢測系統 30 3.2 實驗試劑與化學藥品 31 3.2.1 DNA 檢體 31 3.2.2 聚合酶鏈鎖反應之試劑 31 3.3 實驗步驟 32 3.3.1 檢體解鏈溫度 32 3.3.2 不同檢體之比較 33 第四章 實驗結果與討論 42 4.1 螢光偵測平台測試實驗 42 4.1.1 毛細管內溫度分布之穩定性量測 42 4.1.2 毛細管內溫度分布曲線 43 4.2 不同序列檢體之Tm分析測試結果 43 第五章 結論與未來展望 53 5.1 結論 53 5.2 未來展望 54 參考文獻 55 | |
dc.language.iso | zh-TW | |
dc.title | 毛細管熱對流聚合酶鍊鎖反應產物專一性檢測機制 | zh_TW |
dc.title | Development of a specificity detection mechanism of amplicons by capillary convective Polymerase Chain Reaction | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李達生,王明文 | |
dc.subject.keyword | 毛細管熱對流PCR,解鏈曲線分析,專一性,溫度梯度, | zh_TW |
dc.subject.keyword | Capillary Convection PCR,Melting Curve Analysis,Specificity,Temperature gradient, | en |
dc.relation.page | 56 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-16 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 機械工程學研究所 | zh_TW |
顯示於系所單位: | 機械工程學系 |
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