pcr yield and primer concentration - (Oct/10/2006 )
I would like to clarify some points regarding PCR.....
if i am using forward and reverse primer of 100nM each, given that my taq is good, no. of cycles is enough, dNTP is enough, all the conditions are perfect.... is that the final concentration of my pcr product should not be greater than 2x100nM (ie 200nM)?
my question seems silly but i am wondering if we only get so little pcr product??
please correct me if i am wrong
thanks a lot
Well, if the primers are limiting, then you can get a maximum of 100 nM. Each dsDNA product incorporates BOTH of the primers. But usually primers are not limiting. Other limiting factors can be the enzyme activity (leading to linear rather than exponential amplification for late cycles), template (leading to delayed amplification), or dNTPs, leading to halting of the polymerase reaction. To really understand this, think about some extreme cases, such as making a primer-dimer product (no template, the primers bind only to each other), and long fragment PCR, where the product is 10Kb or more.
and also build up of inhibitive by-products like pyrophosphate and dUTP (from thermal deamination of dCTP)
Primers and polymerase thermostability are not the rate limiting steps in PCR yield.
Deamination is the rate limiter seen beyond 35-40 cycles.
what is meant by deamination?? is it the thermal deamination mentioned by perneseblue?
if yes, then would it mean that increase dNTP concentration can increase the yield of PCR??
Thanks a lot for all your suggestions!!
In real life situations, increasing dNTP concentration (with the accompanying increase in Mg ions) would not increase PCR product yields dramatically. Pyrophosphates inhibition, generation of dUTP -subsequent incoperation-and later polymerase stalling, product inhibition by incomplete melting, polymerase denaturation, etc would be barriers to improved yields.
As there is the real problem of polymerase induced errors, which constantly builds up, and can cause a significant proportion of the product population to contain errors.
It would be better to just increas the number of reaction tubes.