Fidelity of DNA amplification

Thu Apr 20 06:22:15 EST 1995

We have cloned 3000 bp DNA fragment into pUC18 vector and now we are going 
to amplify 2000 bp protein coding sequence out with PCR using 50 bp primers. 
Primers contain 20 bp homological specific sequence and 30 bp unhomological 
tail. Taq DNA polymerase amplify 2000 bp fragment well. 25 ul reaction 
mixture contains for instance 200 uM dNTP, 1.5 or 3.0 mM Mg++, 300 nM 
primers, 1 ng plasmid DNA and 1 U Taq.
Has anyone experience with Pfu, Pwo and Ultma DNA polymerases? Have you 
sequenced your PCR products? How many mismatches have you found? If I use 
one of these enzymes containing 3'-5'exonuclease activity (proofreadig 
activity), what is the probability that I will get 2000 bp ampification 
product without mutations? What kind of conditions should I use? Is it 
better to have less product and combine products from many reactions or 
rather use more cycles, Mg++, dNTPs, DNA or primers? For example with 
Taq DNA polymerase I get more ampified DNA using 3.0 mM Mg++ than 1.5 mM 
Mg++. How important is the dNTP concentration? What is significant 
difference in Mg++ or in dNTP concentrations in DNA ampilification fidelity? 
With Taq DNA polymerase I use 200 uM dNTPs. Is it too much for high fidelity 
DNA amplification? How essential is the quality of the DNA template? 
How about number of cycles, temperatures and other cycling 
parameters? Is there any other proofreading thermostable DNA polymerases for 
PCR? Too many questions? 

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