High fidelity DNA polymerases like "Phusion" have 3'--5' exonuclease activity. What DNA type do they degrade, ssDNA , dsDNA or both? What is the optimal temperate for them, RT, 37 or higher?
This exonuclease activity is also known as proofreading as it removes the incorrectly incorporated nucleotides (mismatches). It also degrades unpaired (therefore single stranded) 3` overhangs. The optimal temperature might differ slightly for each type of enzymes but typically ~70 degrees Celsius.
Hi there,
To go further in Peter's way, Phusion will degrade neither dsDNA nor ssDNA. Its exonuclease activity only occurs on 3' overhangs to generate blunt end and as a proofreading activity eliminating mismatch during extension.
Many people use Phusion DNA polymerase for PCR. If they can degrade DNA, nobody will use it for PCR reactions, because our PCR set up contains DNA template.
I verified the manufacturer's specifications for Phusion. Similarly to other proofreading polymerases, it will degrade primers in the absence of dNTPs, so it should be the last thing you add to your PCR (as should be a habit regardless).
The optimal temperature for its proofreading activity is probably close to its optimal extension temperature but it is most likely significant even at 37 deg C.
Can this enzyme be used in place of T4 ligase for for its exonuclease activity in SLIC cloning?
Both, Phusion and Q5, degraded the PCR product at different temperatures. The results were slightly better when I didn't use the GC-rich buffer. Initially, the product looked like a cloud, but after a few hours, even this cloud disappeared. I used different oligos for ds cDNA MIF-1. When I used KOD polymerase, there was no problem.
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