DNA polymerase fidelity assay (NOT PCR-based) - (Jun/12/2008 )
Hi,
I cloned and purified DNA polymerase I from thermophilic organism, this is not the coomercially available one, such as taq or Pfu. I am planning to characterize the enzyme, I showed this enzyme is active accr to in vitro radiaoactive assay (bu using 3H-dATP and TCA precipitation method).
but I didn't carry out PCR with this DNApolI !!
I want to analyze or test the accuracy (fidelity) of this polymerase , but the all of the assays I searched are on PCR-based, so it is about of the error rate or frequency of DNA polymerase during PCR cycling performed by used polymerase.
is there any assay or experiment , I can perform the accuracy,error rate or fidelity of DNApolI ...
best regards..
I cloned and purified DNA polymerase I from thermophilic organism, this is not the coomercially available one, such as taq or Pfu. I am planning to characterize the enzyme, I showed this enzyme is active accr to in vitro radiaoactive assay (bu using 3H-dATP and TCA precipitation method).
but I didn't carry out PCR with this DNApolI !!
I want to analyze or test the accuracy (fidelity) of this polymerase , but the all of the assays I searched are on PCR-based, so it is about of the error rate or frequency of DNA polymerase during PCR cycling performed by used polymerase.
is there any assay or experiment , I can perform the accuracy,error rate or fidelity of DNApolI ...
best regards..
I am not sure any of these taq purification protocols also contain chekcing fidelity of the purified Taq, but there is no harm chekcing them out, if I were you.
http://search.vadlo.com/b/q?sn=158621799&a...ation&rel=0
..
Maybe you could amplify the same target a number of times, then sequence with a proofreading polymerase to see what error rate you get. If your target DNA is 1kb, you should get some indication of error rate.