CHAPTER 4: Droplet Gene Analysis – Digital PCR
Published:20 Nov 2020
Until recently, quantitative polymerase chain reaction (qPCR) was considered a golden standard for the analysis of nucleic acids, yet the technological advances in microfluidics gave birth to a digital PCR (dPCR) technique that has shaken the analytical landscape. In the dPCR approach, the biological sample is partitioned into a limited but known number of compartments (e.g. wells, droplets, chambers) such that individual (single) target nucleic acid molecules, randomly distributed among compartments, are present either at 0 or 1 copy per single compartment. After the end-point PCR and digital visualization, the partitions containing the DNA molecules will emerge as fluorescent, while negative partitions (containing no DNA) will remain blank. By digitally counting the number of positive partitions, one can precisely estimate the absolute number of target molecules in the sample. In this chapter we focus on a droplet digital PCR (ddPCR) technique that, in contrast to other microfluidics-based systems, provides unmatched scalability and throughput. We discuss various experimental factors that should be considered before conducting ddPCR assays such as fluorophores, surfactants, molecular adsorption and leakage phenomena, template preparation and multiplexing amongst others. We compare three commercial ddPCR systems available to date and present a literature overview of the most important ddPCR applications.