Quantitative poymerase chain reaction (qPCR) is a method to quantify the amplified product in real time by looking at the amount detected at a certain point of the run, as it is directly related to the initial amount of target in the sample.
By using a fluorescent reporter in the qPCR reaction, it is possible to measure DNA generation in the qPCR assay. The linearity of DNA amplification in the assay is used to determine absolute or relative quantities of a known sequence in a sample.
qPCR measures the DNA amplification in real-time while conventional PCR measures the amplification at the end of the reaction.
Step 1: Denaturation: This step uses high heat (around 95°C) to denature the double helix DNA structure into single strands. Step 2: Annealing step uses a lower heat to allow the primers in the reaction mix to bind to the template which is now single stranded. Annealing temperatures are usually around 60°C but can vary depending on the design of the primers. Step 3 is the extension step, where the temperature is again increased to about 72°C to allow for optimal performance of the taq polymerase enzyme which attaches to the site where the primer binds to the template. During this step, the taq moves along the strand incorporating free nucleotides into the sequence to generate a double stranded DNA. Step 4: End of cycle. This is the duration set out by the thermocycler for the extension step. The next cycle starts again from step 1 through step 3 to complete a second cyle. 35 to 40 cycles can be completed for good results.