hello,
can you please tell me if i use the pcr product as a template and do the pcr again so is there any mutation will occur in my 2nd pcr product but as i check the re pcr products by using the gel electro-phorosis the results was good
my question is is there any chance of mutation is pcr product after this.
the rate of misincorporation is constant for a given enzyme and temperature so you will get mutations as you run more cycles but they will be random and of low frequency. The biggest problem with re amplifications is over amplification. Every 10 cycles you will get 1000 times more pcr product so if you are re amplifying then dilute a tiny amount of pcr product ( use 1/10th-100th ul of material) and do not run many cycles otherwise there will be so much product that the product will self anneal and many larger amplification bands will appear. A cleaner way to get more material is to use nested primers where one or both second round primers are located inside the first set of primers. This amplifies a cleaner product although slightly shorter than the original amplimer
the rate of misincorporation is constant for a given enzyme and temperature so you will get mutations as you run more cycles but they will be random and of low frequency. The biggest problem with re amplifications is over amplification. Every 10 cycles you will get 1000 times more pcr product so if you are re amplifying then dilute a tiny amount of pcr product ( use 1/10th-100th ul of material) and do not run many cycles otherwise there will be so much product that the product will self anneal and many larger amplification bands will appear. A cleaner way to get more material is to use nested primers where one or both second round primers are located inside the first set of primers. This amplifies a cleaner product although slightly shorter than the original amplimer
I completely agree with Paul. The advantages of using the PCR product as template in your subsequent PCR is that, if your amplicon is gel purified, then in 2nd round of PCR you can easily eliminate all non specific bands which might have occurred due to regions in your template (may be plasmid) which are complementary to your set of primers. By using the already amplified amplicon as target, you give the reaction no other chances to cross react. The majority of template available is the amplicon. Gel purified PCR product thus serve better purposes here.
As for mutation, I think the incorporation would be same as previous. The rate of incorporation of mispaired bases for one polymerase is fixed. So the mismatches incorporated in the first round would persist and new mispairing would be added on on. But this would normally happen in any PCR cycle irrespective of what template you use.
Also, as Paul mentioned the disadvantages, if your fragment of interest and your template (PCR product) is too small in size, they may self anneal and compete with your primer set for annealing and subsequent amplification. Moreover, I feel primer annealing is better when there are flanking regions like that in plasmid, rather than just the amplicon, where the primer would have to attach at the very end of the template.
Hoep it helps!
there is absolutely no harm in using a PCR product in the second run as a template, as this will give you better (specific) results and mutation should not be a problem.
no problem using a amplicon as template.
if you are concerned about PCR based mutations, use a very high fidelity enzyme, e.g. Phusion or Q5 (NEB). Never use low fidelity enzymes anyway, since even in the first round of PCR many mutations will occur.
Ultimately, you will have to sequence the product anyways to confirm the right identity.
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