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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊曜宇 | |
dc.contributor.author | Jiann-Mou Chen | en |
dc.contributor.author | 陳建謀 | zh_TW |
dc.date.accessioned | 2021-06-13T04:13:44Z | - |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-25 | |
dc.identifier.citation | [01] Laboratory accreditation standards and guidelines for nucleic acid detection techniques., National pathology accreditation advisory council.
[02] K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, H. Erlich., “Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.,” Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73. [03] C. Oste., “Polymerase chain reaction.,” Biotechniques. 1988 Feb;6(2):162-7. [04] P. M. Holland, R. D. Abramson, R. Watson, and D. H. Gelfand., “Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.,” Proc Natl Acad Sci U S A. 1991 August 15; 88(16): 7276–7280. [05] J.A. Brainard, J.K. Greenson, C.J. Vesy, R.J. Tesi, A.C. Papp, P.J. Snyder,L. Western, T.W. Prior., “Detection of cytomegalovirus in liver transplant biopsies. A comparison of light microscopy, immunohistochemistry, duplex PCR and nested PCR.,” Transplantation. 1994 Jun 27;57(12):1753-7. [06] R. Novati,V. Thiers,A.D. Monforte,P. Maisonneuve,N. Principi,M. Conti, A. Lazzarin,C. Brechot., “ Mother-to-child transmission of hepatitis C virus detected by nested polymerase chain reaction.,” J Infect Dis. 1992 Nov;166(5):1192-3. [07] G. M. Severini, L. Mestroni, A. Falaschi, F. Camerini and M. Giacca., “Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples.,” J Clin Microbiol. 1993 May; 31(5): 1345-1349 [08] J. Skeidsvoll , P.M. Ueland., “Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.,” Anal Biochem. 1995 Nov 1;231(2):359-65. [09] T. Morris , B. Robertson , M .Gallagher., “Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system.,” J Clin Microbiol. 1996 Dec;34(12):2933-6. [10] http://www.appliedbiosystems.com.tw/product/product_3-2-2.htm [11] J. Compton., “Nucleic acid sequence-based amplification.,” Nature. 1991 Mar 7;350(6313):91-2. [12] Dos Remedios C.G., M. Miki , J.A. Barden., “Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.,” J Muscle Res Cell Motil. 1987 Apr;8(2):97-117. [13] V.T. Förster., “Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann.Phs.,” 1948. 2: p.55-75 [14] A. A. Deniz, M. Dahan, J. R. Grunwell, T. Ha, A. E. Faulhaber, D. S. Chemla, S. Weiss, and P. G. Schultz., “Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations.,” Proc. Natl. Sci. USA Vol. 96, pp. 3670-3675 March 1999 [15] G. J. Schütz, W. Trabesinger, and T. Schmidt., “Direct Observation of Ligand Colocalization on Individual Receptor Molecules.,” Biophys J, May 1998, p. 2223-2226, Vol. 74, No. 5 [16] T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok., “Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.,” FEBS Letters 531 (2002) 245-249 [17] M. Kim, C. V. Carman, T. A. Springer., “Bidirectional Transmembrane Signaling by Cytoplasmic Domain Separation in Integrins.,” Science 301 (5640): 1720-1725 [18] R. M. Siegel, J. K. Frederiksen, D. A. Zacharias, F. KM. Chan, M. Johnson, D. Lynch, R. Y. Tsien, M. J. Lenardo., “Fas Preassociation Required for Apoptosis Signaling and Dominant Inhibition by Pathogenic Mutations.,” Science 30 June 2000: Vol. 288. no. 5475, pp. 2354 – 2357 [19] http://www.finnzymes.fi/products/rt-pcr/amv_reverse_transcriptase.htm [20] http://www.ebiotrade.com/buyf/productsf/epicentre/relation/hybridase.PDF [21] http://www.neb.com/nebecomm/products/productM0251.asp [22] http://www.sigmaaldrich.com/sigma/datasheet/R0884dat.pdf [23] http://www.roche-applied-science.com/pack-insert/0881767a.pdf [24] B. Deiman, Pv. Aarle, and P. Sillekens., “Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA).,” Molecular Biotechnology, February 2002, Volume 20, Issue 2, pps. 163-180 [25] A. Heim, I.M. Grumbach, S. Zeuke and B. Top., “Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA) .,” Nucleic Acids Res, 1998, Vol. 26, No. 9 2250–2251 [26] G Leone, H van Schijndel, B van Gemen, FR Kramer and CD Schoen., “Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA.,” Nucleic Acids Res, 1998, Vol. 26, No. 9 2150–2155 [27] S. Kimura, K. Fujinaga,T. Sekiya., “PCR machine.,” Tanpakushitsu Kakusan Koso. 1996 Apr;41(5):514-7. [28] S. Tyagi, K. Bhol, K. Natarajan, C. Livir-Rallatos, C. S. Foster, and A. R. Ahmed., “Ocular cicatricial pemphigoid antigen: Partial sequence and biochemical characterization.,” Proc. Natl. Acad. Sci. USA,Vol. 93, pp. 14714-14719, December 1996 [29] http://mekentosj.com/science/fret/index.html [30] R.E. Dale, J. Eisinger, and W.E. Blumberg. 1979., “The orientational freedom of molecular probes.,” Biophys. J. 26: 161-193 [31] http://www.invitrogen.com [32] http://www.olympusfluoview.com/applications/fretintro.html [33] J.R. Lakowicz., “Principles of Fluorescence Spectroscopy.,” Vol. 1. 1999, New York: Kulwer Academic/Plenum Publishers [34] G. W. Gordon,G. Berry, X. H. Liang, B. Levine, and B. Herman., “Quantitative Fluorescence Resonance Energy Transfer Measurements Using Fluorescence Microscopy.,” Biophysical Journal 1998.Volume 74 May 2702–2713 [35] A. R. Clapp, I. L. Medintz, and H. Mattoussi., “Forster Resonance Energy Transfer Investigations Using Quantum-Dot Fluorophores.,” ChemPhysChem 2006, 7, 47–57 [36] J. J. Li., “Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA.,” Nucleic Acid Res, 2000.28(11): p.e52 [37] H. Y. Li, E. K. On Ng, S. MY. Lee, M. Kotaka, S. K. W. Tsui , C. Y. Lee, K. P. Fung, M. M.Y. Waye., “Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET).,” Journal of Cellular Biochemistry,2000 Vol. 80, (3) , pp. 293 – 303 [38] W. Gloffke., “Reviewing the latest trends and applications in quantitative, real-time PCR.,” The Scientist 2003, 17(8):41 [39] P. Nupam. Mahajan, L, Katrina, B. Gail, W. G. Gordon, R. Heim and B. Herman., “Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer.,” Nature Biotechnology 16, 547 - 552 (1998) [40] S.Angers, A. Salahpour and ¬M. Bouvier¬., “DIMERIZATION: An Emerging Concept for G Protein–Coupled Receptor Ontogeny and Function.,” Annual Review of Pharmacology and Toxicology 2002 Vol. 42: 409-435 [41] B. Jacques. de Kok , E.T.G. Wiegerinck, B. A.J. Giesendorf, D. W. Swinkels., “Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).,” Human Mutation 2002 Vol. 19,(5) , pp. 554 – 559 [42] A. Solinas, L. J. Brown, C. McKeen, J. M. Mellor, J. Nicol, N. Thelwell and T. Brown., “Duplex Scorpion primers in SNP analysis and FRET applications.,” Nucleic Acids Res, 2001, Vol. 29, No. 20 e96 [43] Y. P. Sher, J. Y. Shih, P. C. Yang, S. R. Roffler, Y. W. Chu, C. W. Wu, C. L. Yu,and K Peck., “Prognosis of Non–Small Cell Lung Cancer Patients by Detecting Circulating Cancer Cells in the Peripheral Blood with Multiple Marker Genes.,” Clinical Cancer Research, Vol. 11, 173–179 January 1, 2005 [44] http://biochem.roche.com/lightcycler/ [45] A. Reiser, M. Geyer, R. van Miltenburg, M. Nauck, and K.Tabiti., “Mutation Detection Using Multi-color Detection on the LightCycler System.,” Biochemica 1999 No.2 , pp.1-15 [46] http://www.invitrogen.com [47] S. S. Wong, E. Joselevich, A.T. Wooley, C. L. Cheung and C. M. Lieber., “Covalently functionalized nanotubes as nanometer-sized probed in chemistry and biology.,” Nature 1998 Vol. 394 pp.52-55 [48] P. D. Morris and K. D. Raney., “DNA Helicases Displace Streptavidin from Biotin-Labeled Oligonucleotides.,” Biochemistry, 38 (16), 5164 -5171, 1999 [49] Z. Yin, J. Severin, M. C. Giddings, W. Huang, M. S. Westphall, L. M. Smith., “Automatic matrix determination in four dye fluorescence-based DNA sequencing.,” Electrophoresis 1996, 17, 1143-1150 [50] L.G.. Lee, K.J. Livak, B. Mullah, R. j. Graham, R.S. Vinayak and T.M. Woudenberg., “Seven-Color, Homogenous Detection of Six PCR Products.,” BioTechniques 27:342-349 (August 1999) [51] R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger, C. A. Mirkin., “Selective Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical Properties of Gold Nanoparticles.,” Science, Vol 277, Issue 5329, 1078-1081 , 22 August 1997 [52] D. J. Zhou, J. D. Piper, C. Abell, D. Klenerman, D. J. Kang and L. Ying., “Fluorescence resonance energy transfer between a quantum dot donor and a dye acceptor attached to DNA.,” Chem. Commun., 2005, 4807–4809 [53] Y. X. and P. E. Barker., “Semiconductor nanocrystal probes for human metaphase chromosomes.,” Nucleic Acids Res. 2004; 32(3): e28. [54] Y. J. Liu, R. C. Ruaan , K. Y. Hsu., “Quantum Dots Surface Modification and Bio-Conjugation.,” Master thesis, Department of Chemistry Engineer, Chung Yuan Christian University,Taiwan 2005 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32700 | - |
dc.description.abstract | 本研究發展出一種具備快速即時性、高準確性、高靈敏度之恆溫特定RNA序列偵測,更對此方法建構出造價低廉的核酸序列檢測儀器。
在本研究中指出使用恆溫RNA 擴增進行特定RNA 基因序列擴增,在90 分鐘內利用1 ng total RNA 將特定基因(TRIM28)放大至 108 倍以上,證實實驗具有相當高的靈敏度。進而將恆溫RNA 擴增與螢光能量共振轉換(Fluorescence Resonance Energy Transfer,FRET)方法做結合,進行即時性的螢光訊號偵測,藉此判斷特定RNA 序列的存在。為求提高增加實驗之準確性,特別將探針增加一組,個別標記有FRET 效應之不同螢光分子,必須兩個探針皆正確的鍵結在序特定列上,才視為真正偵測到特定基因序列,藉此增加其正確鍵結的困難度,以求得即時性、高準確性、高靈敏度之恆溫特定RNA 序列偵測。 實驗結果顯示,本論文成功地結合恆溫RNA 擴增與FRET,開發出具備即時性、高準確性、高靈敏性之核酸序列檢測方法。 由於市售之量子點顆粒過大使得FRET 並沒有顯著表現。並且進一步進行量子點的表面改質,其結果穩定性不夠,也無法應用於本研究中。 因此本論文所設計之激發光源與儀器,寄望未來量子點的顆粒能縮小,且具備更穩定的型態,使得本研究發展之具備即時性、高準確性、高靈敏度之即時性恆溫特定RNA 序列偵測,能夠在靈敏度達到更進一步。 | zh_TW |
dc.description.abstract | In this thesis, we develop a detecting method with high accuracy and high sensitivity for specific RNA sequences in isothermal environment. We construct a low-cost and real-time system for detection specific RNA sequences by using this method.
Isothermal RNA amplification is a powerful tool to amplify specific sequences of RNA. It can successfully amplify as low as 1 ng of total RNA in 90 minutes. Therefore, we integrate isothermal RNA amplification and Fluorescence Resonance Energy Transfer (FRET) methods to real-time detect fluorescent signals for specific RNA sequences. For increase specificity, two hybridization probes which can bind to the same gene are used in our experiments to increase the accuracy. Base on this study, a novel method with higher accuracy and sensitivity for detecting specific RNA sequence in isothermal environment can be achieved. The results shown that the FRET signal of TRIM28 and UCHL1 genes have been detected in 40-60 min. Therefore, we have successfully setup a new low-cost, real-time detection system, based on combined Isothermal RNA Amplification and FRET. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T04:13:44Z (GMT). No. of bitstreams: 1 ntu-95-R93921128-1.pdf: 2275507 bytes, checksum: 4e12c220e204c99d8ac2d8280e97f48e (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 致謝--------------------------------------------------------------------------------------------- I
中文摘要-------------------------------------------------------------------------------------- II 英文摘要------------------------------------------------------------------------------------- III 目錄------------------------------------------------------------------------------------------- IV 圖目錄--------------------------------------------------------------------------------------- VII 第一章 緒論---------------------------------------------------------------------------------- 1 1-1 、核酸檢測簡介------------------------------------------------------------------- 1 1-2 、研究動機與目的---------------------------------------------------------------- 4 1-2.1、恆溫RNA 擴增---------------------------------------------------------- 4 1-2.2、螢光共振能量轉換------------------------------------------------------ 5 1-3 、本文概觀------------------------------------------------------------------------- 6 1-4 、貢獻------------------------------------------------------------------------------- 7 第二章 理論分析---------------------------------------------------------------------------- 9 2-1 、核酸序列擴增------------------------------------------------------------------- 9 2-1.1、恆溫RNA 擴增介紹---------------------------------------------------- 9 2-1.2、恆溫RNA 擴增之設計要點------------------------------------------- 9 2-1.3、恆溫RNA 擴增流程---------------------------------------------------10 2-1.4、恆溫RNA 擴增應用---------------------------------------------------12 2-2、螢光共振能量轉換--------------------------------------------------------------13 2-2.1、螢光原理-----------------------------------------------------------------13 2-2.2、螢光共振能量轉換原理-----------------------------------------------14 2-2.3、Förster resonance energy transfer 方程式演算--------------------16 2-2.4、螢光共振能量轉換效益-----------------------------------------------18 2-2.5、螢光共振能量轉換應用-----------------------------------------------22 第三章 實驗設計---------------------------------------------------------------------------23 3-1 、實驗架構設計-----------------------------------------------------------------23 3-2 、螢光分子核酸探針設計-----------------------------------------------------24 3-2.1、螢光分子選擇-----------------------------------------------------------24 3-2.2、螢光分子核酸探針設計-----------------------------------------------25 3-2.3、螢光量子點探針設計--------------------------------------------------26 3-3 、硬體系統設計-----------------------------------------------------------------27 3-4 、激發光源設計-----------------------------------------------------------------28 3-4.1、基板-----------------------------------------------------------------------28 3-4.2、汞燈座--------------------------------------------------------------------28 3-4.3、濾片座-------------------------------------------------------------------------29 3-4.4、聚焦鏡片座--------------------------------------------------------------29 3-4.5、光纖座--------------------------------------------------------------------29 3-4.6、封裝之前蓋--------------------------------------------------------------30 3-4.7、封裝之風扇蓋-----------------------------------------------------------30 3-4.8、封裝之機殼--------------------------------------------------------------30 3-4.9、激發光源組合-----------------------------------------------------------30 3-5 、螢光光譜訊號分析-----------------------------------------------------------31 3-5.1、光譜訊號分離處理-----------------------------------------------------31 第四章 實驗材料、設備與方法---------------------------------------------------------39 4-1、光學系統組件------------------------------------------------------------------39 4-1.1、激發光源-----------------------------------------------------------------39 4-1.2、激發光源電源供應器--------------------------------------------------39 4-1.3、激發光源濾片-----------------------------------------------------------39 4-1.4、光譜儀--------------------------------------------------------------------40 4-1.5、收光透鏡-----------------------------------------------------------------40 4-1.6、溫度調變比色管支撐器-----------------------------------------------40 4-1.7、光纖-----------------------------------------------------------------------41 4-1.8、光譜儀操作軟體--------------------------------------------------------41 4-2、引子與探針設計---------------------------------------------------------------41 4-3、生物實驗------------------------------------------------------------------------42 4-3.1、細胞培養----------------------------------------------------------------42 4-3.2、細胞Total RNA 萃取-------------------------------------------------42 4-3.3、RT-PCR(將RNA 反轉錄為cDNA)-----------------------------43 4-3.4、以PCR 反應擴增特定核酸序列------------------------------------44 4-3.5、以PCR 反應驗證螢光探針鍵結------------------------------------45 4-3.6、恆溫RNA 擴增--------------------------------------------------------45 4-3.7、恆溫RNA 擴增即時螢光偵測--------------------------------------46 4-3.8、DNA 洋菜凝膠電泳 ( DNA Agarose Gel Electrophoresis) -----46 4-3.9、恆溫RNA 擴增產物洋菜凝膠電泳) -------------------------------47 4-4、量子點探針(Quantum Dot Probe)製備----------------------------------47 4-4.1、Quantum Dot Streptavidin Conjugates DNA Probe 合成--------47 4-4.2、Quantum dot Thiol Probe 合成---------------------------------------48 4-4.2.1、Quantum dot 表面改質------------------------------------------48 4-4.2.2、Quantum Dot Thiol Probe 鍵結--------------------------------49 4-5、Förster 半徑之計算-------------------------------------------------------------50 4-6、FRET 螢光光譜分析-----------------------------------------------------------50 第五章 實驗結果與討論------------------------------------------------------------------52 5-1、激發光源之建構---------------------------------------------------------------52 5-1.1、收光系統之分析--------------------------------------------------------52 5-1.2、激發光源組合-----------------------------------------------------------55 5-1.3、硬體系統組合-----------------------------------------------------------56 5-2. 訊號處理驗證--------------------------------------------------------------------57 5-3. 恆溫RNA 擴增實驗驗證-----------------------------------------------------59 5-4. 即時性的恆溫特定RNA 序列偵測驗證------------------------------------61 5-4.1、探針雜合反應之可行性驗證-----------------------------------------61 5-4.2、使用PCR 確定引子與探針之FRET 之可行性------------------66 5-4.3、即時性的恆溫特定RNA 序列偵測---------------------------------67 5-4.3.1、即時性的恆溫特定RNA 序列偵測I ---------------------------67 5-4.3.2、即時性的恆溫特定RNA 序列偵測II --------------------------70 5-5、量子點的應用驗證------------------------------------------------------------72 5-5.1、Quantum dot 之靈敏度優勢驗證------------------------------------72 5-5.2、Quantum dot 探針之雜合反應與FRET 驗證----------------------73 5-5.3、表面改質後之Quantum dot 探針螢光表現------------------------75 5-6、討論------------------------------------------------------------------------------77 第六章 結論與未來展望------------------------------------------------------------------80 6-1 、結論-----------------------------------------------------------------------------80 6-2 、未來展望-----------------------------------------------------------------------81 第七章 參考文獻---------------------------------------------------------------------------83 附錄 ------------------------------------------------------------------------------------------87 | |
dc.language.iso | zh-TW | |
dc.title | 利用螢光共振能量轉換與恆溫RNA擴增之快速即時核酸偵測系統 | zh_TW |
dc.title | A Rapid and Real-time Nucleic Acid Detection System by Fluorescence Resonance Energy Transfer and Isothermal RNA Amplification | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊泮池,孫啟光,蔡孟峰 | |
dc.subject.keyword | 螢光共振能量轉換,恆溫RNA擴增,核酸偵測, | zh_TW |
dc.subject.keyword | Fluorescence Resonance Energy Transfer,Isothermal RNA Amplification,Nucleic Acid Detection, | en |
dc.relation.page | 89 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-25 | |
dc.contributor.author-college | 電機資訊學院 | zh_TW |
dc.contributor.author-dept | 電機工程學研究所 | zh_TW |
顯示於系所單位: | 電機工程學系 |
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