A versatile oscillating-flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis.

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.