Digital PCR is a relatively new approach to nucleic acid detection and quantification that offers an alternate method to conventional real-time quantitative PCR for absolute quantification and rare allele detection. It all works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions; some of these reactions contain the target molecule (positive) while others do not (negative). A single molecule can be amplified a million-fold or more. During amplification, TaqMan chemistry with dye-labeled probes is used to detect sequence-specific targets. When no target sequence is present, no signal is emitted. Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without the need for standards or endogenous controls.
That’s how it all works on paper, but seeing it in person is a different game altogether. The workflow for the QuantStudio 3D digital PCR system was recently updated and posted on Thermo Fisher Scientific’s YouTube channel. Take a look at the workflow in the video below, and let us know if you have any questions in the comments section.