Endpoint PCR, quantitative PCR and digital PCR
Here are the dynamics of the reaction plotted as cycles number versus the amount of synthesized PCR product. In the exponential phase of the reaction, there is a theoretical doubling of amplicons with each cycle. In later cycles, approximately cycles 30 to 35, there may be depletion of critical reagents such as dNTPs, which slows down or stops PCR amplification. This is referred to as the plateau phase. Assessments in traditional endpoint PCR are often made in the plateau phase. Endpoint PCR, quantitative PCR, and digital PCR all include the same reaction dynamics, but the way the PCR products are analyzed is different. With endpoint PCR, the end products are visualized on an agarose gel to determine their size as well as relative quantity. Endpoint PCR is used for applications such as cloning, sequencing, genotyping and sequence detection. Endpoint PCR is far less quantitative than real-time PCR—it is used mostly to detect presence or absence of targets, but can also be used to estimate relative quantity. This determination is made at the end of temperature cycling. In quantitative PCR, or qPCR, the PCR products are continuously detected throughout the reaction cycles via either intercalating or probe-based fluorescent dyes. The fluorescent signal increases proportionally as product amount increases. qPCR is much more accurate in quantifying the starting material than endpoint PCR. Using a standard curve generated by amplifying known amounts of starting DNA, the quantity of target DNA can be determined from the cycle number at which the unknown DNA is detected relative to control DNA. qPCR, as part of RT-qPCR, can be used to quantitate RNA levels; the first step converts RNA to single- stranded cDNA. Primary qPCR applications include gene expression profiling, copy number variation, molecular diagnostics, and genotyping. Digital PCR is the only “absolute” quantitative PCR method available, in that it measures the actual number of target molecules produced as opposed to comparing the measurement to a reference.