This is becuase;
1. DNA polymerase after 30-35 cycles is usually denatured, becuse each denaturation temp (95-94) for 30 sec to 1 min effects the protein function and it is not tolerable after 35 cycles usually.
2. If DNA polymerase even have the activity, as some new generation recombinantly engineered DNA polymerase has, there will be more chances for errors.
3. as Prakash Chandra said, exausting dNTPs could be the case, Can you run further PCR cycles after addition of only dNTPs? I dont think it is possible.
One reason is that you dilute the concentration of the primers over time (because the primers are integrated into the newly synthesized strand) and the template concentration increases (exponentially). This imbalance between template and primers may lead to the template out-compeeting the primer during the annealing phase therefore the reaction becomes inefficient. Also your polymerase fidelity decreases over time and the number of mutations increases.
after 60 to 70 cycles the enzyme is burn out you will have an incresed of Taq mutations in the DNA and unspecific amplifications!
usually you do a nested or a Tri-nested PCR, one PCR with 30 to 4o cycles and them another 1 or 2 pcr with the same number of cycles. between PCR's you will dilute your sample 1/10 to 1/1000 from the prevoius PCR to reduce the unspecific amplification.
more reason could be that the amount of DNTPs used.. they are used by polymearse for synthesis of amplicons..
This is becuase;
1. DNA polymerase after 30-35 cycles is usually denatured, becuse each denaturation temp (95-94) for 30 sec to 1 min effects the protein function and it is not tolerable after 35 cycles usually.
2. If DNA polymerase even have the activity, as some new generation recombinantly engineered DNA polymerase has, there will be more chances for errors.
3. as Prakash Chandra said, exausting dNTPs could be the case, Can you run further PCR cycles after addition of only dNTPs? I dont think it is possible.
And because, with each cycle, you increase the risk of unwanted mutations due to the fidelity of the polymerase. None have 100% fidelity.
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