Polymerase chain reaction (PCR) has been modified by three generations. The first generation of PCR relies on gel electrophoresis to analyse PCR products, but is challenged by low detection limit, laborious operation, and single application (qualitative). The second generation of PCR (also called real-time quantitative PCR (RT-qPCR)), can quantify the products with a standard curve, but also show low tolerance to interfering substances. Digital PCR (dPCR) is the third generation of PCR that enables absolute quantification through partitioning the reaction. Highly sensitive and accurate in molecular detection, this technology has demonstrated applications like trace DNA detection, rare mutation detection and copy number variation.
Digital PCR, droplet PCR or digital droplet PCR as it is also called, quantifies the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. Digital droplet PCR is particularly useful for needle-in-a-haystack detection of genes such as the detection of residual leukemia cells in the bone marrow of patients during morphologic remission.
To understand why traditional PCR is limiting in such scenarios, it is important to understand what happens during a PCR reaction. A basic PCR run can be broken up into three phases:
Exponential: exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. Exponential amplification occurs because all of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.
Linear (high variability): as the reaction progresses, some of the reagents are being consumed as a result of amplification. The reactions start to slow down and the PCR product is no longer being doubled at each cycle.
Plateau (End-point: gel detection for traditional methods): the reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade. Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is where traditional PCR takes its measurement, also known as end-point detection.
Traditional PCR measures at the plateau, giving you variable results
Real-time PCR measures at the exponential phase for more accurate quantitation
Digital PCR counts individual molecules for absolute quantification
(Information sources: Mao et al., Principles of digital PCR and its applications in current obstetrical and gynecological diseases, Am J Transl Res. 2019; 11(12): 7209-7222. and Thermo fisher Scientific).
Quantitation of targets for PCR by use of limiting dilution by P J Sykes 1, S H Neoh, M J Brisco, E Hughes, J Condon, A A Morley
pubmed.ncbi.nlm.nih.gov/1389177/
This video is about Droplet Digital PCR, ddPCR protocol, Digital PCR technology
Quantitative PCR, ddPCR vs. qPCR, DNA quantification methods, Molecular diagnostics
Digital PCR applications, High-throughput PCR, Absolute quantification, Rare mutation detection, Copy number variation analysis, Gene expression analysis, Liquid biopsy applications and PCR workflow optimization.
15 май 2023
