I want to amplify a 1500bp amplicon, when I use pfu DNA polymerase the PCR product size is 1000bp, when I use taq DNA polymerase it is 1500 bp. Do somebody have some comments in this regard?
Most people attribute this phenomenon to either extension times that are too short for Pfu (which is less processive than Taq), hence favoring shorter amplicons that arise from non-specific primer binding, or to the Pfu chewing back primers in the absence of dNTPs if components have been added to the reaction mix in the wrong order (Taq cannot do it).
I don't buy either explanation, though, as I've seen cases where this happens regardless of extension time or proper experimental manipulation, so I'd be interested too in other people's insight!
The difference between buffers of the Taq DNA Polymerase and pfu DNA Polymerase may effect the electrophoretic mobility of the PCR products. One suggestion is that you can purify the PCR proudcts, sequence them and conduct the sequence alignment.
you can increase the extension time in the cycle as well as in the final extension and also increase the ddNTPs concentration.
Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand.
I agree with Alejandro Martín explanaton, the proofreading activity of pfu can sometimes shortened the primers that can bond in a most favourable time. I will increase the annealing temperature if primers are highly epecific and increase the extension time. How much did you include in your PCR program??
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