在DNA確定為人類的遺傳重要因子後，歐美各國皆將其視為重點發展的科技，其中又以Real Time PCR最受囑目，這是因為Real Time PCR能夠對DNA作定性及即時定量，檢測人體是否染有病毒。Real Time PCR能做即時定量，是因為DNA加入了探針(Probe)且具有螢光偵測系統，故螢光偵測系統是定量技術的關鍵所在。本研究嘗試開發一套螢光偵測系統，能運用在DNA的螢光偵測當中，螢光檢測的核心採用Cooled CCD。由於增生後的DNA所散發出來的螢光相當微弱，加上CCD元件本身會發熱而產生熱雜訊(暗電流效應)，使得照出來的螢光和雜訊難以分別，加上激發光源以及實驗環境週邊亦會有雜光的產生，且熱雜訊、雜光皆會影響實驗的結果，故熱雜訊及雜光的消除就格外重要。因此利用致冷晶片、散熱鰭片及風扇所搭配之致冷技術將CCD熱雜訊消除，成為Cooled CCD，並先對HBV DNA濃度為 108 copies/ml且加入螢光試劑SyBr Green 之樣本進行拍攝，確認雜訊是否濾除，其後採用HBV DNA濃度為 108 copies/ml且加入螢光試劑Red 670之樣本進行拍攝，彌補SyBr Green專一性不足且無法做multiplex的缺點。另外以Trace Pro光學軟體模擬採用黑體輻射噴漆與濾鏡後，能有效的降低雜光對實驗的影響。並再最後分析溫度與螢光偵測之間的關係。

Through fluorescent monitoring at each cycle of the polymerase chain reaction (PCR), real-time PCR has been the powerful investigative tool for DNA quantification. In this study, a cooled CCD was developed for the fluorescence detecting system of the real time PCR machine. The 1600(H) x 1200(V) interline transfer progressive scan monochrome CCD chip (Kodak, KAI 2020M) is employed for the high sensitive detector. The 2 million pixels sensor consists of 7.4 μm square pixels with microlenses can provide the output gain to 20mV/e- by a three-stage source-follower amplifier. The binning mode operation can extend the full well capacity to 80,000 e-. By a two-stage thermoelectric cooler, the chip is cooled down to -20oC. The advantages of the cooled CCD include low noise, high sensitivity and high resolution. That yields a powerful solution for DNA fluorescence signal analyzing and quantification. The scattering light induced interference for the fluorescence detection was analyzed by the Trace Pro software. Based on the simulation results, a blackbody spray painted chamber was designed for enclosing the DNA sample and the detection system. The SyBr Green labeled DNA fluorescence images could be captured with high signal to noise ratio. Moreover, the Red 670 labeled fluorescence images were recorded. The poor excitation efficiency of this special dye yields ultra low intensity images. Although the signal to noise ration of red 670 fluorescence images is 20.5dB, we still can clarify DNA templates exist or not in the sample by image analyzing.

摘 要 i
ABSTRACT ii
誌 謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1前言 1
1.2研究動機與目的 2
1.3文獻回顧 3
第二章CCD影像感測器 5
2.1影像的基本概念 5
2.2 CCD影像感測器動作原理 6
2.2.1光電轉換 6
2.2.2電荷的儲存 7
2.2.3電荷的傳輸 9
2.2.4電荷的檢測 11
2.3 CCD影像感測器的特性 12
2.3.1靈敏度 12
2.3.2 動態範圍 13
2.3.3 解析度 14
2.3.4雜訊 15
第三章 致冷系統 16
3.1熱電效應 16
3.1.1 Seebeck 效應 16
3.1.2 Peltire 效應 17
3.2 致冷晶片原理及特性 18
3.3 致冷晶片的選用 21
3.3.1 致冷晶片尺寸規格 21
3.3.2 實驗上致冷片的選用 23
第四章 微生物實驗 26
4.1 DNA的結構與複製原理 26
4.1.1 DNA的結構 26
4.1.2 DNA的複製原理 30
4.2 Real Time PCR 35
4.2.1 Real Time PCR的介紹 35
4.2.2 Roche LightCycler 38
4.3 DNA 螢光檢測 41
4.3.1螢光原理 41
4.3.2 螢光檢測方式 43
第五章 Trace Pro光學軟體模擬 45
5.1 Trace Pro 光學軟體 45
5.2 Trace Pro 光學軟體模擬 46
5.3模擬結論 52
第六章 研究設備與實驗步驟 53
6.1 Cooled CCD 的開發之研究設備與實驗步驟 53
6.1.1 Cooled CCD 之研究設備 53
6.1.2 Cooled CCD 之實驗步驟 57
6.2 DNA螢光拍攝(Red 670)之研究設備與實驗步驟 60
6.2.1 DNA螢光拍攝(Red 670)之研究設備 60
6.2.2 DNA螢光拍攝(Red 640)之實驗步驟 68
第七章 實驗結果與分析 72
7.1 Cooled CCD 開發之實驗結果分析 72
7.1.1 冷卻溫度結果與溫差分析 72
7.1.2 雜訊抑制結果與分析 73
7.2 DNA螢光拍攝(Red 670)之實驗結果分析 75
7.2.1 螢光試劑差異分析 75
7.2.2 溫度控制對螢光檢測之結果分析 76
第八章 結論 83
參考文獻 85

[1]J. Watson and F. Crick, "A structure for deoxyribose nucleic acid," Nature, vol. 171, 1953, pp.737.
[2]J. W. Jorgenson and K. D. Lukacs, "Zone electrophoresis in open-tubular glass capillaries," Anal. Chem, vol. 53, 1981, pp.1298-1302.
[3]K. B. Mullis, F. Ferre and R. A. Gibbs, "The polymerase chain reaction," Birkhauser, 1994.
[4]C. Kim, "Charge transfer in charge-coupled devices," J. App. Ph., Vol. 42, no.9, 1971, pp.3586-3594.
[5]W. S. Boyle and G. E. Smith, "Charge Coupled Semiconductor Device," Bell Syst. Tech. J., Vol. 49, 1970, pp.587-593.
[6]米本和也原著，陳榕庭，彭美柱譯，CCD/CMOS 影像感測器之技術與應用，台北，全華科技圖書股份有限公司，2005。
[7]J. E. Carnes, "Free charge transfer in charge-coupled devices," IEEE Trans. Electron Devices, ED-19, 1972, pp.798-808.
[8]W. F. Kosonocky, "Charge Coupled Devices - An Overview," Western Electronic Show and Convention Technical Papers, Vol. 18, 1974, pp.2/1-2/20.
[9]T. R. Hsu, MEMS and Microsystems：Design and Manufacture, McGraw-Hill Inc., 2002.
[10]K. Chen and S. B. Gwilliam, "An analysis of the heat transfer rate and efficiency of TE(Thermoelectric) cooling systems," International Journal of Energy Research, vol. 20, 1996, pp.399-417.
[11]I. Kim and D. Lee, "An analysis for geometrical effects on the cooling performance of (Bi, Sb)2 Te3 / Bi2(Te, Se)3 - based thin film thermoelectric modules," Materials Research Society, vol. 12-2, 1997, pp.423-429.
[12]R. Venkatasubramanian, B. O’Quinn, E. Siivola, K. Coonley, M. Napier and P. Addepalli, "Nano-scale Thermoelectric Materials for Solid State Cooling and Direct Thermal to Electric Energy Conversion", Nature, vol. 413, 2001, pp.597.
[13]Melcor,http://www.melcor.com/
[14]分子生物學總論，http://www.supermt.com.tw/edu/molbio/lecture_1/1204.pdf
[15]陽明大學基因體科學研究所，http://www.dls.ym.edu.tw/lesson/dna.htm
[16]D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry 3rd Ed., Worth Publishers, 2000.
[17]馬階醫院，http://www.mmh.org.tw/taitam/patho/SERVICE/molecular/PCR
[18]台灣醫學感染管制學學會，http://www.nics.org.tw/magazine/16/02/16-2-7.htm
[19]M. C. Tseng, Detection of genetically modified soybean by semi-quantitative and real-time PCR, Master, National Chung Hsing University, Taichung, 2004.
[20]P. M. Holland, R. D. Abramson, R. Watson and D. H. Gelfand, "Detection of specific polymerase chain reaction product by utilizing the 5'' to 3'' exonuclease activity of thermus aquaticus DNA polymerase," Proc Nat Acad. Sci. USA, vol. 88, 1991, pp.7276-7280.
[21]R. Alary, A. Serin, D. Maury, J. P. Sirven, M. F. Gautier and P. Joudrier, "Comparison of simplex and duplex real-time PCR for the quantification of GMO in maize and soybean," Food Control, vol. 13, 2002, pp.235-244.
[22]G. Dolino, D. Bellet and C. Faivre, "Adsorption strains in porous silicon,"
Physical Review B, vol. 54, no. 24, 1996.
[23]Dvcco,http://www.dvcco.com/PDFs/datasheets/imagers/KAI-2020LongSpec.pdf
[24]Sigma-Aldrich, http//www.sigmaaldrich.com/Local/SA_Splash.html