Continuous flow digital PCR for absolute quantification of nucleic acids

peer-reviewedDigital polymerase chain reaction (PCR) has emerged as an extremely powerful technique for quantification of nucleic acids. Digital PCR offers superior quantification accuracy, precision and sensitivity compared to quantitative PCR – the current gold standard in nucleic acid quantification. The quantification capabilities of digital PCR permit new applications such as non-invasive prenatal diagnosis and rare mutation detection in cancer diagnostics. The technique also offers improvement in viral load quantification for HIV monitoring and residual minimum disease quantification for monitoring disease progression. Digital PCR quantifies the number of targets by partitioning a biological sample into many individual reactions, performing PCR and directly counting the number of positive reactions in an experiment. Quantification is highly accurate and precise since the starting quantity of target molecules is not derived from standards or Cq values as with quantitative PCR. As digital PCR quantification is absolute, very small copy number ratios can be precisely measured. The thousands of reactions used in a digital PCR experiment permits single molecule sensitivity, a fundamental characteristic of digital PCR. The commercially available digital PCR instruments employ microfluidic arrays or droplets to partition samples into thousands of individual reactions. These instruments require expensive consumables, greatly increasing the experimental cost and limiting experimental design. Continuous flow PCR is a novel technology that generates, thermal cycles and fluorescently detects droplets in a continuous system that does not require any consumables - facilitating low cost digital PCR. A continuous flow digital PCR instrument was designed and developed at the Stokes Institute. These droplet based instruments operate by delivering droplets through the temperature zones required for PCR. The flowing droplets are fully wrapped and separated by an immiscible carrier fluid that prevents contamination on the device. Two droplet production techniques were characterised. Liquid bridge dispensers were employed in this thesis as they were shown to produce highly consistent droplets as small as 45nL in volume. Carryover contamination at the liquid bridge dispenser was identified and countered using wash steps. A proof of concept amplification study demonstrated the devices capability of amplifying a DNA target in a flowing droplet. A GAPDH and RNase P target was amplified on the device in two separate experiments and verified using gel electrophoresis. Digital PCR was successfully performed on the continuous flow platform. This is the first time absolute quantification using digital PCR has been performed in a flowing system. Droplets were stable, highly consistent and results demonstrated that there was no cross over contamination on the instrument. Amplification of a single RNase P target molecule in a flowing droplet was achieved, demonstrating single molecule sensitivity. Singleplex digital PCR was used to quantify various concentrations of RNase P target molecules in gDNA, ranging from 178 – 6100 copies/mL. The singleplex quantification results correlated extremely well with the theoretical copy number calculations. Duplex digital was performed on the instrument, examining the copy number ratio of RNase P to SRY. The digital copy number ratio quantification results deviated from the expected RNase P to SRY ratio of 2:1. A quantitative PCR study confirmed that this was due to poor optimisation of the duplex assay and was not related to instrument performance. Continuous flow instruments are a highly desirable alternative to current digital PCR platforms. The technology is best placed to perform digital PCR for applications requiring quantification of rare targets in large sample volumes. The flexibility in droplet production permits processing of large sample volumes quickly and at a low cost versus commercial digital PCR platforms which require numerous consumable chips