Quantitative Polymerase Chain Reaction Using Infrared Heating on a Microfluidic Chip

摘要

The IR-mediated polymerase chain reaction (IR-PCR) in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of the IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR long-pass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 templates at different starting copy numbers were amplified, and the threshold cycle showed an increase for decreasing amounts of starting DNA. The amplification efficiency was 80percent, and the gel separation indicated no detectable nonspecific product. A melting curve was generated using IR heating, and this indicated a melting temperature of 85 deg C for the 304 bp amplicon, which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification, and in highly integrated systems that combine pre- and post-PCR processes.