The Study of Slide-based PCR Thermal Cycler
Date Issued
2013
Date
2013
Author(s)
Huang, Chung-Che
Abstract
Real-time quantitative polymerases chain reaction (real-time qPCR) is combined DNA amplification and fluorescent sensing for pathogen detection. Because laboratory instruments with high sensitivity and low detection are usually bulky, the concept of portable real-time qPCR is developed. This research is focused on the miniaturization of PCR thermal module. We select thin silicone film as heating unit and used PDMS as PCR chip. This PMMA-based device is constructed out by CNC machining, and it is modularized compactly in order to accommodate silicone film, fan, heat sink and thermocouple. Heating and cooling rate of the heating module is 1.1 and 0.7°C/s separately and the temperature amplitude is smaller than 0.1°C. In order to suit the dark-field fluorescent sensing module, the height of reaction chamber is 1mm and the chamber contains 10μl per reaction. Taq polymerase is apt to absorb on the surface of PDMS, 2.5% PVP is added in PCR mixture to avoid absorption and it doesn’t inhibit polymerase activity. We use the cDNA sequence of H1N1 as target template. In order to improve PCR efficiency, we choose 50 °C as annealing temperature. In home-made heating module authentication, commercial PCR instrument is a control group. The fluorescent signal is analyzed by sigmoid model fitting to the amplified efficiency, and the efficiency were found to be 0.97 (commercial instrument) and 0.70 (homemade). If we can integrate dark-field fluorescent sensing module, a portable real-time qPCR instrument will achieve the concepts of on-site detection or point-of-care.
Subjects
即時定量聚合酶鏈鎖反應
矽膠加熱片
PCR晶片
H1N1
Type
thesis
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