A Novel Contamination Free Two Temperature Continuous Flow Polymerase Chain Reactor

摘要

Polymerase Chain Reaction (PCR) is an enzymatic process that has dramatically advanced many fields of life sciences, where it is an indispensable tool in a burgeoning range of applications, including diagnostic medicine, molecular biology, forensics and food testing. Recent increased demand for extremely high throughput PCR systems has led to the development of miniaturised continuous flow microfluidic PCR devices, which may have extremely high throughput compared to standard commercial PCR thermal cyclers. A novel continuous flow microfluidic PCR device has been designed and fabricated, consisting of two thermal zones maintained on aluminium thermal blocks providing the precise temperatures required for denaturation and annealing/extension. Polycarbonate sideplates retain the denaturation thermal block vertically above the annealing/extension thermal block while allowing for a variable air gap to be maintained between them. Heating of the denaturation thermal block is achieved using a Labview controlled Thermofoil heater, while the annealing/extension thermal block is maintained at temperature by optimised heat transfer from the denaturation block. Flow-through capillary tubing is positioned into a grooved serpentine channel machined into these thermal blocks. This serpentine channel passes through each thermal block fifty times, providing fifty PCR thermal cycles. Contamination free high throughput continuous flow PCR necessitates that the samples be encapsulated in an immiscible carrier fluid to eradicate cross contamination between samples and suppress the likelihood of the sample contacting the capillary leading to sample degradation. Encapsulation of the PCR reaction mixture is achieved upstream of the thermal cycler through segmentation of the sample into droplets entrained within an immiscible carrier fluid, which are then cycled through the thermal cycler. High throughput DNA amplification of two genes, GAPDH and LEF1, from the REH cell line has been successfully demonstrated on this microfluidic platform without any detectable contamination between samples. The PCR droplet reactors were approximately 250nl which is two orders of magnitude less than the standard sample size for most commercial PCR thermal cyclers.