以往復式熱循環系統進行反轉錄聚合酶連鎖反應

Integrated oscillatory thermocycler for reverse transcription polymerase chain reaction

林宗宏(Zong-Hong Lin)

指導教授 ： 陳志堅

國立屏東科技大學/工學院/生物機電工程系所/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00291

摘要

本論文以往復式熱循環系統進行反轉錄聚合酶連鎖反應，使用CFDRC進行溫控模組加熱塊之熱傳模擬。在不同加熱塊高度、反應槽大小與加熱棒數目，探討加熱速度與熱平衡時間的影響。從而設計出適合的加熱塊。本研究主要是通過自製加熱器、溫控模組與機電定位模組來完成RT-PCR。而RT-PCR溫控主要分為RT與PCR兩個部分做溫控，通過移動反應槽於自製溫控模組三個溫區間，藉由反應槽的移動，控制其速度、停留位置與停留時間來滿足RT-PCR所需的溫度條件。溫控模組主要依中溫區、高溫區與低溫區排序分佈，通過機電定位模組移動反應槽於高溫區進行加熱，而降溫則是把反應槽從高溫區移動到低溫區進行降溫。設定較低的低溫區溫度，使當高溫區進入低溫區時因為較大的溫差能夠快速地降溫。從而避免使用致冷晶片快速降溫後導致溫度過低，當溫度過低的同時導致需要花費較多的時間進行升溫。因為此種溫區佈置，從而減少反應槽從低溫升到中溫所需的時間。因為當反應槽從低溫區往中溫區移動的同時會先經過高溫區，因為經過高溫區，反應槽在經過高溫區的同時也被高溫區進行加熱，因此當抵達中溫區的，反應槽的溫度已經不需要中溫區在進行加熱，只需維持反應槽的溫度。最終裝置完成RT-PCR反應擴增出285bp的片段。系統加熱塊溫度的均勻性在2K以內。反應槽往復速度皆為24mm/s，進行40個熱循環，反應時間為2小時5分，與對照組相比有99.7%的擴增效率，樣本最少量為10μl。

關鍵字

反轉錄聚合酶連鎖反應 ； 往復式 ； 熱循環裝置

並列摘要

This paper used a reciprocating thermocycler to conduct a reverse transcription polymerase chain reaction (RT-PCR), and a CFDRC to conduct a heat transfer simulationonthe heating blocks of a temperature control module. On heating blocks of different height, reaction vessel size and heating rod number, measurements of rate of heating and thermal equilibrium time were conducted. From there, a suitable heating block was designed. This study primarily made use of a self-made heater, temperature control module and electromechanical positioning module to complete the RT-PCR.The temperature control action of the RT-PCR thermostat was primarily shared between the RT and PCR by moving the reaction vessel through three temperature zones within the self-made heat control modules. The movement of the reaction vessel, including the speed, rest position and duration of rest, was controlled in order to satisfy the necessary temperature conditions of the RT-PCR. The temperature control module was arranged with mid, high and low temperature zones.An electromechanical positioning module was used to move the reaction vessel to the high temperature zone to create heating; while for cooling, the reaction vessel was moved from the high temperature zone to the low temperature zone. Setting a lower temperature in the low temperature zone made it so that when moving from the high temperature zone to the low temperature zone, the larger temperature spread would allow for a quick decrease in temperature. Also,the arrangement avoids the overcooling that results from using a cooling wafer to rapidly reduce the temperature, as well as the extra time and cost necessary to bring the temperature back up once it has been overcooled. Due to the temperature zone arrangement, the time needed for the reaction vessel to come from a cool temperature to a mid-temperature is reduced. When the reaction vessel moves from the low temperature zone to the mid-temperature zone, it first passes through the high temperature zone and increases its temperature, therefore when it arrives in the mid-temperature zone the temperature merely needs to be maintained, rather than increased. In the end, the apparatus completed the RT-PCR amplification of the 285bp fragments. System heating block temperature uniformity within 2K. Reciprocating speed the reaction vessel are all 24mm / s, for 40 thermal cycles, the reaction time was 2 hours and 5 minutes, as compared with the control group 99.7% efficiency of amplification, the sample least volume was 10μl.

並列關鍵字

Reverse transcription polymerase chain reaction ； Oscillatory ； Thermal Cycler

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