We have amplified a slightly large segment of K562 cDNA -2271bp- in E.Coli TOP10. At first, we’ve used of MaxTaq Enzyme (Vivantis Com.), but the mutation rate was so great and we found 5 Homozygote mutations. After that, we changed the Enzyme type and we applied the Hi-fid DNA polymerase, as a result, we have identified 3 Homozygote mutations in this stage. It’s interesting that, there were not any shared mutations between these two groups. Before cloning this region as we were not sure about cell line and enzyme fidelity this region was sequenced and we did not face any variation. What’s the problem in your opinion? As you know, we applied several various stages for screening and pureeing the DNA fragment. I don’t know what’s the problem and which step has problem!!
How can I reduce these mutation’s rate? What’s your suggestion?
Use Phusion polymerase or KOD polymerase that are best available high-fidelity enzymes. We have amplified using these polymerases up to 7 Kb with no mutations.
Use Phusion polymerase or KOD polymerase that are best available high-fidelity enzymes. We have amplified using these polymerases up to 7 Kb with no mutations.
I would recommend phusion as well.
In addition you can increase the amount of template DNA, if too little template DNA is added and a mutation occurs in the first couple of cycles the mutation will be propagated to a larger proportion of the final product molecules, howevere with a higher number of initial template molecules there will be a greater number of first round product molecules and with a high-fidelity polymerase its highly unlikely that a mutant product arising later will become a significant proportion of the whole number of amplified molecules. In other words use more template and fewer cycles of amplification.
Please use Q5 (NEB) or Pfu turbo (Agilent) DNA polymerases that normally used for amplifying whole plasmid in site-directed mutagenesis. They are high-fidelity DNA polymerases and therefore you can reduce mutation rates during PCR amplification by using them.
I second the ideas above of using KOD (Hot-Start) polymerase (Kamal Kumar) and increased template/fewer cycles (Martin Craig Taylor) based on my experience. I have cloned a few dozen ORFs this way and whenever there was a sequence difference in my clone that differed from the published sequence, I found that it was the sequence of the template that was different and not a mutation introduced by PCR. I tried to limit my PCR to 10 - 15 cycles.
I can't tell from your question if you are sequencing your amplified products directly or subcloning them first. It sounds like the latter in that you are "screening" the DNA fragment but I'm not sure exactly of your workflow.
My point is that if you are subcloning the PCR product, then the host strain, especially if it is different than the original TOP10 host, may be introducing mutations because it doesn't like the cDNA for some reason. I have had that happen as well: I PCR up an ORF, confirm the product by direct sequencing, and then find point or deletion mutations when sequencing the clones. If you're cloning from TOP10 to TOP10 then I don't know why that would be happening. If you sequence and confirm the amplicon before introducing it into a plasmid and E. coli host, then you can look for a different strain to work in. Good luck!
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