In the annealing step, why would NOT the two original strands of DNA rejoin together, and thus inhibit the reaction? How the concentrations of both the original DNA and the primers are related to this matter?
In PCR all possible types of pairing take place. And since in a PCR reaction, you dont have a single DNA molecule and just one cycle, so there is no inhibition, as you describe it.
it was just an assumption for that matter, Mr. Abhijeet Singh do you think there is any relationship between the molar excess of primers vs target and the annealing of DNA not to happenhappen in PCR?
As you say in pcr the template which is very long will re anneal at high temperature and this will have an effect on pcr efficiency in the first few cycles. However, the primers are in huge molar excess compared with the template so the melted template strands will spend much time looking for an antisense template to re anneal with but the primers will immediately find a binding site because the rate of reaction is dependent on the concentration of the 2 molecules and as the template is a fixed amount the rate of reaction is then dependent only on the concentration of the primer which is very high. So even though the primers anneal at a lower temperature they do bind much more efficiently at the annealing temperature. if the gradient of temperature was very slow from 94 to 60 then the template might reanneal but at the fast movements between temperatures the primers will get their chance to anneal
- Badges
- Science method