Proof Reading Taq Polymerase - How effective can it be in mutation study (Aug/31/2005 )
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The ordinary Taq DNA polymerase has no 3' to 5' exonuclease activity and is known for its misincorporation. When one wants to study muatations in a gene of a particular bacteria ( by sequencing the PCR product say 450 bp to 750 bp), can the ordinary Taq be used for the PCR or should a proof reading Taq be used? Will the ordinary Taq be able to give accurate amplified products? since even a single misincorporation may mislead us. What are the challenges in optimizing the reaction when a proof reading Taq is used?
No polymerase will guarantee 100% correct amplification. If you want to reduce your chances of having "false positives" (thanks to mutations incorporated by taq) you can use a proofreading enzyme. There's a lot of them around, just check the manufacturer's websites and choose the one that's best for you.
On the other hand, if you do some math, you can check the chances of having specific mutations in your gene thanks to taq. If you are looking for just 1 or 2 specific mutations (i guess your mutation matters mostly if another amino acid is incorporated, right?), you have only 6 bp to care about. Then think about the fact that PCR is an exponential process, meaning that if say your first 3 cycles went without a problem (i.e. without the specific mutations on the 6 bp in the case I'm explaining) and taq incorporates 1 bp change in a strain, you have only 1/16 mutations in your final PCR product. (I oversimplify of course). So for this purpose, i think regular taq might do just fine. If you want to detect any mutation, then definately go for proofreaders.
You can check it btw, just do your PCR several times (5 times, 10 times), starting from the same template, and check for differences in the sequence... (they should in theory be thanks to PCR-errors).
On the other hand, if you do some math, you can check the chances of having specific mutations in your gene thanks to taq. If you are looking for just 1 or 2 specific mutations (i guess your mutation matters mostly if another amino acid is incorporated, right?), you have only 6 bp to care about. Then think about the fact that PCR is an exponential process, meaning that if say your first 3 cycles went without a problem (i.e. without the specific mutations on the 6 bp in the case I'm explaining) and taq incorporates 1 bp change in a strain, you have only 1/16 mutations in your final PCR product. (I oversimplify of course). So for this purpose, i think regular taq might do just fine. If you want to detect any mutation, then definately go for proofreaders.
You can check it btw, just do your PCR several times (5 times, 10 times), starting from the same template, and check for differences in the sequence... (they should in theory be thanks to PCR-errors).
I would only use taq for cloning if I knew I could sequence the resulting product to ensure fidelity (Primrose, Twyman, and Old; 2003). Taq maintains its fidelity the same way Bill Clinton did: not particularly well.
I would use something like Accuprime Pfx (BEST) or Pfu (a little cheaper) in place of Taq.
-Matt