A Real-Time Continuous Flow Polymerase Chain Reactor for DNA Expression Quantification

Abstract

Real-time quantitative Polymerase Chain Reaction (PCR) is an extremely sensitive and reliable method for quantifying gene expression, allowing subtle shifts in gene expression to be easily monitored. Currently, stationary real-time PCR is readily achieved using fluorescent labels which increase in fluorescence as the DNA is exponentially amplified. Quantitative PCR is used in a myriad of applications. However currently most commercial real-time PCR devices are batch process stationary well based systems, limiting their throughput. Continuous flow microfluidic PCR devices have allowed for advancement in terms of improved PCR throughput and reduced reagent usage. As part of an overall total analysis system a device integrating all the functional steps of continuous flow realtime quantitative PCR has been designed and fabricated. Initially the PCR reaction mixture is segmented into nano-litre PCR reactors which are then thermally cycled on a two temperature fifty cycle flow-through PCR device, which allows laser induced fluorescent imaging of the nanoreactors. Previous studies into continuous flow PCR have demonstrated endpoint fluorescent measurements, however this research allows PCR nanoreactors to be fluorescently monitored after every PCR thermal cycle. Fluorescent optical monitoring is achieved through laser excitation of the nanoreactors while a Charged Coupled Device (CCD) camera is used to record the fluorescent emissions from the nanoreactors. Intensity analysis of the recorded images is then preformed using MATLAB to accurately determine the fluorescence intensity level, thereby allowing real-time quantitative amplification curves to be generated. This has major advantages over existing continuous flow PCR devices which use endpoint fluorescence and capillary electrophoresis, as the amplification curves allow far more information to be gleaned and allow the initial DNA template concentration to be accurately determined.