Real-Time Fluorescence Monitoring of the Polymerase Chain Reaction in a Novel Continuous Flow Reactor for Accurate DNA Quantification

Abstract

Real-time Polymerase Chain Reaction (PCR) is the preferred method for quantification of gene expression levels due to its extreme sensitivity. Fluorescence based real-time PCR is commonly used for the quantification of the initial amount of a specific sequence of DNA. Real-time quantification may be achieved using fluorescent dyes, by optically monitoring the product formation as the PCR cycles progress. Stationary well based real-time quantification is quite common, however continuous flow real-time PCR which is the aim of this work is still in its infancy. A compact, high throughput continuous flow thermal cycler has been developed which allows for real-time fluorescent measurements to be obtained. The principle of operation of this device is that the three thermal zones required for a polymerase chain reactor are maintained on both sides of an aluminium block and bio-compatible FEP Teflon capillary tubing is then wrapped around these constant temperature blocks. The capillary tubing is wrapped around the device fifteen times which provides thirty PCR thermal cycles. The device has been designed and optimised to accurately monitor the product expression level using the double stranded DNA binding dye SYBR green I. Initially the PCR mixture is segmented into small nanoreactors, separated by an immiscible carrier fluid to eliminate cross contamination and reduce the likelihood of sample degradation due to contact with the capillary wall. These PCR nanoreactors are then cycled through the tubing and the DNA amplified. Fluorescent optical monitoring of these nanoreactors takes place where a water glycerine mixture, which is refractive index matched to the tubing, allows for improved fluorescent measurements of the nano-volume reactors to be obtained. Plasmid DNA, 240 base pairs long, has been successfully amplified using this device and the temperatures for the denaturation, annealing and extension phases have been accurately measured. Real-time fluorecence images of the flowing nano-volumes were recorded every second cycle using a CCD camera and from these images amplification curves have been successfully generated. Samples with various initial concentrations of DNA have been thermally cycled on the continuous flow reactor. The measured increase in fluorescence intensity from the flowing nano-volume reactors as they progressed through the thermal cycler demonstrated the effect of initial DNA template concentration.