- 學位論文
溫控機制設計及其用於核酸定量
The Design of Temperature Control Mechanism and Its Function on Nucleic Acid Quantification
摘要
Real Time PCR 技術，為近年來最重要的分子生物學進展，該技術可快速在單一載具多個樣本裡針對多種病原體 DNA 得到定性以至於定量的分析，改進了傳統技術耗時、容易污染以及不方便性。實現此技術的儀器，主要需結合溫控機構與螢光檢測儀，在溫控過程進行 DNA 體外增生，同時監控過程中 DNA 嵌合螢光的多寡，進行 DNA 分析。 本論文最主要是承蒙恩師開發之 Real Time PCR Machine ，將溫控機制調整至最佳化並應用於核酸定量上。在機台的升降溫控機制方面本研究提出一套 PID 控制理論，以 Visual Basic 寫成控制程式，控制風扇轉速的快慢，來控制吹進反應室裡空氣的高低溫，以達升降溫之目的，使 PCR 實驗能穩定地增生成功。再藉由洋菜凝膠電泳與紫外光吸收檢測系統，進行產物分析，並與 ABI 9700 PCR Machine做實驗對照組，驗證機台的穩定性及可靠度。由實驗結果得知本研究所研發之溫控技術，可成功應用在核酸定性上，目前已完成103 copies/ml sample增生，並且在高成功機率下，成功增生108、106 copies/ml sample。 在螢光檢測系統上，因為增生反應後的 DNA 所散發出來的螢光相當微弱，且螢光偵測元件本身會發熱而產生暗電流效應，使得照出來的螢光強度和熱雜訊難以分別，加上激發光源以及實驗環境週邊亦會有雜光的產生，易影響實驗的結果，故熱雜訊及雜光的消除就格外重要。於此，吾人自行設計光路結構，且採用有 Cooled 功能的螢光偵測元件，以降低熱雜訊對螢光訊號強度的影響，並透過訊噪比分析得知，本研究架設出的螢光檢測儀，其螢光測試的動態範圍，下限為1 fmol ，且可分辨無讀值之水。而目前本研究已初步達成 DNA 標記螢光偵測，並能有效收集頻譜數據，用於定量分析，未來將就其量測可靠度進行評估，以利技術能迅速落實於業界產品。
並列摘要
This Real Time PCR machine allows the detection of DNA amplification through out the detection of the fluorescence labeling dye in the PCR mix during the early phase of this reaction. In addition, the concentration of target DNA fragment in the PCR mix before thermal cycling can be obtained from the time recorded history of the fluorescence intensity by integrating thermal cycler and fluorimeter. The Real-Time PCR machine has higher sensitivity and consumes less time than those of the traditional PCR machine. This study modified the temperature control mechanism developed by the previous studying results for the optimization of DNA amplification. A novel PID control program coded by Visual Basic was proposed to achieve optimized control of thermocycling of PCR. By 2% Agarose gel electrophoresis and UV absorption detection system, the yield products from this novel machine and the ABI 9700 PCR instrument were compared to verify the stability and reliability of the machine developed by our team. This results show that the temperature control scheme proposed in this study can be successfully applied in nucleic acid qualitative analysis. The 103 copies / ml initial copies sample has been successfully amplified. The 108 and 106 copies / ml samples were amplified successfully with high reproducibility. Regarding to the fluorescent detection system, a novel optical design was also proposed in this study. Because of the very weak fluorescent from the labeling dye attached on the DNA double strands, the fluorescent detection device itself should have very low dark current and the environmental light interferences should be avoided. The optical structure with black body painting surface was constructed to adopt cooled CCD based fluorescent detection devices to reduce the noise of fluorescent signal detection and isolate the sample under detection for high signal to noise ratio measurement of the fluorescence during PCR. The detection limit of this fluorescent detection system is 1 fmol Fluorescein. The signal can be resolved with the readings of the water. To summarize, this study has constructed a Real Time PCR machine for DNA amplification and quantification. In the future, we this instrument can be commercialized for a low cost and high accuracy DNA quantification solution.