單一溫控熱對流聚合酶連鎖反應系統 之開發與研究

Abstract

單一溫控熱對流聚合酶連鎖反應系統之開發與研究 謝一帆 國立台灣大學機械工程研究所

中文摘要 熱對流聚合酶連鎖反應 ( CPCR，Convective Polymerase Chain Reaction )的概念首先於2002年被提出，主要是指控制兩個加熱器溫度的不同造成溫度梯度而產生熱對流現象，並可成功進行PCR增生。本文則針對此熱對流PCR系統進行改良，提出---單一溫控之熱對流PCR系統，並就如何成功增生DNA片段進行研究。本實驗主要是使用玻璃毛細管承載75μL之反應試劑，配合恆溫槽與油浴機制加熱，藉此控制毛細管底部為反應樣本之變異作用溫度 ( 94℃)，因試劑表面接觸室溫而冷卻產生熱對流現象。同時利用粒子影像測速儀 ( PIV，Particle Image Velocimetry )，PIV觀測毛細管內流場分佈與速度，測得底部到頂部之循環所需時間，藉此得到PCR反應之最佳參數。也利用熱電偶與紅外線測溫儀 ( IR，Infrared Spectroscopy ) 兩種系統，量測毛細管內部溫度分佈，以分析流場循環過程中是否達到PCR三階段的溫度。本實驗也嘗試利用Fluent模擬，在此邊界及初始條件下之流場及溫度分佈的情形，並與實驗量測進行比對。根據本實驗結果，於25分鐘後，B型肝炎病毒之質體核酸（30 copies/tube）122-bp之 DNA 成功地專一性擴增出來。 本實驗證明可利用一簡單架構---單一熱源之恆溫槽與油浴加熱方式，成功進行熱對流PCR增生反應，並探討圓柱體之毛細管內單一熱源之熱對流溫度和流場分佈，搭配其PCR實驗結果，發現流場速度快慢與PCR成功機率有顯著性的關係。藉由此數據的建立，相信可對往後即時定量檢測熱對流PCR建立有極大助益。

Development of a Novel Convective Polymerase Chain Reaction System with Single-Temperature Control Hsieh, Yi-Fan Department of Mechanical Engineering, National Taiwan University, Taipei, Taiwan, R.O.C.

ABSTRACT The concept of convective PCR (Convective Polymerase Chain Reaction) was revealed in 2002. The convection is generated by the temperature gradient controlled by a couple of heaters. This essay focuses on the improvement of the design of the heater – single heating source. We provided a glass capillary tube of 75μL used as the vessel, combined with constant temperature control in the oil-bath. The temperature of the bottom of the capillary tube is kept 94℃ for denaturing. The surface of the capillary tube is cooled by contacting the room temperature. Thus the convection observed is an effective method for amplification of the oligo-nucleic acid and the detection of the mutation and infected disease. By using the particle image velocimetry, PIV method, to observe the convection in the tube, the flow and the velocity distribution are obtained. Meanwhile, the convection from the bottom to the top of the tube can be inspected to calculate the optimized parameters for PCR. Thermal couples and IR ( Infrared Spectroscopy ) are also employed for the inspection of the temperature distribution of the capillary tube. The simulated results calculated by using the commercial suite Fluent is also compared with the observed PIV results. Since the experimental results show that the 122-bp plasmid of hepatitis B virus of 30 copies/ tube can be specifically amplified successfully, we can conclude that the velocity of the fluid is strictly related to the probability of the successful PCR. This research proves that the convection caused by a single heater can successfully execute PCR. Due to the conclusions of this essay, we believe that it is helpful to establish the real-time convective PCR.