It is possible because the DNA polymerase is also active at temperatures other than 72*C (or whatever you'd have chosen for extension). So, there is extension as soon as the primer anneals although the rate of polymerization might be slower.

What you describe is commonly known as two-step PCR.

Hope it helps!

It is possible because the DNA polymerase is also active at temperatures other than 72*C (or whatever you'd have chosen for extension). So, there is extension as soon as the primer anneals although the rate of polymerization might be slower.

What you describe is commonly known as two-step PCR.

Hope it helps!

Hi Gyanendra,

in addiction to what said Alejandro Martin , the 2-step PCR you described means that Tm for annealing primers need to be between 72 and the denaturation temperature (92-95°c).

I would advise you to redesign the primers to get a lower Tm since 2-step PCR may lack of efficiency and specificity. obviously, a Tm between 55 and 60°c should be better.

fred

Could you mentioned the pcr profile? Sometimes there are no strict rules for temperature setting and your primer + polymerase can work above 72 effectively. Your amplicon size is also small that could be a reason too.

As well as polymerases being active over a wide temperature range as Alejandro Martin said on the way back up to denaturing temperatures the reaction will pass through 68-72c and with polymerases extending at between 100 and 1000 bases a second (depending on which polymerase you use) there is plenty of time for amplimer production as most pcr machines ramp at 4c/second or slower

I agree, polymerases are active over a wide temperature range and do their job (maybe not adequately and correct but they do) in your reaction (steps) .

Thank you all for your valuable suggestions.