Why are yields always lower with high-fidelity DNA polymerases (proofreading enzymes)?
Because these high-fidelity DNA polymerases have the 3’ to 5’ exonuclease proofreading activity to degrade single-stranded DNA. The primers are single-stranded DNA which means that the specificity designed into primer can be changed during the course of the PCR. However, that is not the only time when high-fidelity DNA polymerase (proofreading activity) contributes negatively in PCR. At the end of each cycle, after the DNA polymerase has finished extension and you have a completed duplex, the proofreading activity can remove the last few incorporated nucleotides and then because there is dNTP present, the polymerase activity repair the nucleotide back in. The problem comes at the end of extension when you heat to denature the duplex you have generated. You denature, and you now have a hyperthermophilic polymerase with an inherent proofreading activity and the ends of your PCR product have been denatured. Until it is cooled down and primer has bound, that is during that temperature tran
