Terminating PCR: How to Control DNA Amplification Length

[nokia8650]

I have a question regarding PCR which I was unable to find the answer to (maybe because its obvious and I am just not thinking of the reason!) How is PCR terminated? For instance, say you wish to amplify a 2kb length of dna, how does one ensure that just this 2kb region is amplified as opposed to a 100kb region for instance - how do you make sure that the polymerase doesn't just keep copying the template strand in a given cycle. I know that there are the denaturation, annealing and elongation phases, but I just don't understand how the elongation phase is terminated at the right point.

Thanks

 

[Borek]

What role primers play?

 

[nokia8650]

primers determine the 5' start of the new strand... not the 3' end?

 

[Borek]

Do you use, or two primers? Why?

Think what is length of a copies from the second & third DNA generation. To simplify things imagine you are throwing away all earlier generations before starting next cycle.

That is not exactly the case, but it doesn't matter much.

 

[nokia8650]

I started to reply about how i still didnt understand, but then as I tried explaining why I saw the reason! Although the polymerase may go beyond the region of interest, on the next cycle, the primer will only bind to the start of the region of interest, and so after a few cycles, youll be left with just the region of interest.

Thanks so much!