What is PCR overcycling? How do I know if my product is overcycled?
What is PCR overcycling? How do I know if my product is overcycled?
PCR overcycling is when cycling goes beyond the exponential phase of amplification. Overcycling occurs when the following events take place during PCR:
-Depletion of substrates (dNTPs or primers).
-The reagents (dNTPs or enzymes) are no longer stable at the denaturation temperature.
-The PCR polymerase is inhibited by the product (pyrophosphate, duplex DNA).
-Competition for reagents (dNTPs and primers) by nonspecific products.
-Lowering of the pH of the reaction.
-Incomplete denaturation/strand separation of products at high product concentrations.
The indicator of PCR overcycling is an intense background smear with indistinguishable bands when the reaction is resolved on an agarose gel. It is always recommended to perform a preliminary test to determine the minimal number of PCR cycles needed to yield a sufficient product. The PCR product remains in the linear phase of amplification if the product yield is noticeably increased every 3–5 cycles. We find that overcycled cDNA does not produce suitable template for any downstream application.