Real-time microchip polymerase-chain-reaction system

A real-time micro-polymerase chain reaction (μ-PCR) system was developed to monitor the amplification of the complementary DNA (cDNA) molecules of the hepatitis C virus (HCV). This system uses SYBR Green I dye as a fluorescent probe for real-time detection in the PCR process. The μ-PCR chip was fabricated on a silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding. Compared to the polypropylene tube used in conventional PCR equipment, the silicon reaction well, which has a high thermal conductivity, can improve the temperature uniformity of the sample and the desired temperature can be attained more rapidly. The closed-loop thermal cycling system consists of power supplies, a thermal generator, a computer-controlled PID controller, a data acquisition subsystem, and a fluorescent microscope. Fluorescence was monitored at the end of the 72°C step of the PCR cycle at five-cycle intervals. This μ-PCR system enables in situ detection and achieves 35 thermal cycles in 26 min. Different initial concentrations of HCV cDNA were used and good performance of this μ-PCR system was verified. Using this real-time μ-PCR system, it is possible to achieve DNA amplification with real-time detection within 26 min.