In hybridation step and after decreasing temperature from 95 to 50-60°C, why does the single-stranded DNA bind to primers and does'nt bind another time to its complementary DNA?
Hi ..
During the hybridization step, the temperature is typically decreased from 95°C to 50-60°C to allow the primers to bind to the target DNA. At these lower temperatures, the complementary base pairing between the primers and the target DNA is more stable, allowing the primers to bind specifically to the target DNA.
Once the primers have bound to the target DNA, the temperature is increased again to around 72°C to allow the DNA polymerase enzyme to extend the primers and synthesize new DNA strands. The new DNA strands are complementary to the template DNA, and therefore do not bind to the primers again. Instead, they are used as templates for the synthesis of additional copies of the target DNA.
After the extension step, the temperature is typically increased to around 95°C again to denature the DNA, separating the two strands of the double-stranded DNA (dsDNA) template so that they can serve as templates for the synthesis of additional copies of the target DNA. This cycle of denaturation, annealing, and extension is repeated multiple times to amplify the target DNA.
The primers are in excess in the reaction and the two strands of the template molecule can "drift" away from each other due to random movement.
As Katie A S Burnette said the primers are in large molar excess. The rate of reaction (binding) depends on the molar concentration of the interacting molecules. The high concentration of primer will drive the primer annealing to template much faster than the relatively rare template reannealing with template molecules. After many cycles of pcr the amount of double stranded product does become comparable to the amount of primer and then product reannealing unevenly can lead to some unexpected bands and smears in a pcr reaction
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