Study of real-time polymerase chain reaction in Rayleigh-Benard convection

• Main Authors: Hsing-Ta Wu, 吳欣達
• Other Authors: Ming-Jyh CHERN
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/57755469628994632995

碩士 === 國立臺灣科技大學 === 機械工程系 === 98 === This study aims to develop the Real-Time polymerase chain reaction (Real-Time RB-PCR) system at a Rayleigh-Bernard convection reactor. SYBR Green I, a fluorescent dye, is used to visualize the double stranded DNA. Natural convection driven by a hot bottom and a cold lid at an enclosed cylindrical container is employed to transport DNA to various thermal environments where three steps of PCR can be performed. A variety of boundary temperatures, dimensions of cylindrical tubes, and viscosities of fluids are considered to obtain the optimal parameters for RB-PCR experiments. To understand the effect of natural convection on the Real-Time RB-PCR system, numerical simulation are undertaken to obtain thermal distribution at the cylindrical container. In addition, the natural convection inside the container is measured using the Particle Image Velocimetry (PIV). After RB-PCR experiments, electrophoresis experiments are also performed to investigate the products of PCR. From the experimental results, it is found that there exists a significant relationship between the stability of flow field and the efficiency of PCR reaction. For example, fluids with a higher viscosity can improve the stability of natural convection at the container. However, the momentum of natural convection is an important factor to determine whether DNA can undergo the three thermal levels of PCR. To clarify the difference among three temperature levels, a group of fluorescence dyes are employed. Therefore, we can immediately know the situations including denaturation, annealing, and extension at the RB-PCR container by the distribution of fluorescence. Also, we can effectively obtain the brightness curve line. It is also found that each process of a PCR cycle is not accomplished only with one single convection loop. To duplicate the target DNA, a few of convection loops are necessary. Results of this study may make more innovations in a RB-PCR reactor.