Science Buddies: "Ask an Expert"

I understand how PCR works. The DNA gets denatured and becomes two ssDNA. The forward primer attaches itself to one strand and the reverse primer attaches itself to another.

My question is how does the primer know when to stop synthesising?

For example, let say our double stranded DNA sequence is this:


5' GTTTTTTTTTTTAAAAAAAAAAG3'
3' CAAAAAAAAAATTTTTTTTTTTC5'

So the forward primer would be say this: 5'GTTTTTTT3'
The reverse primer is: 5'GAAAAAA3'

Now, when the dsDNA denatures and become two ssDNA, the primers will attach. However, wouldn't the primer just keep on synthesising forever? Besides from the annealing time expiring, how does the primer know when to stop?

At first my answer to this question was the use of stop codons...but the codons are actually used during translation and are not important in the actual PCR?

Thanks for your help.

Sorry, just wanted to add something to the original post.

My question concerns what happens if you have primers that suppose to be specific for a gene within the DNA template.

I know that if the goal was just to amplify as much of the original DNA template, then the primer would just continue synthesising until it reaches the end of the DNA. However, if we are trying to PCR a specific gene within a huge DNA template, how does the primer know when to stop?

Thanks

Hi,

... ABOUT PCR ...

The polymerase does not know when to stop. It may synthesize hundreds or thousands of extra bases before either: 1) the polymerase just falls of naturally which happens frequently which makes it hard to synthesize very long pieces of DNA by PCR or; 2) the cycle time on the thermocycler ends and the temperature goes up to over 90 degrees and the extra heat causes the polymerase to fall off.

This does not matter. The extra long DNA synthesis will only happen with the original template DNA which goes on a long way in both directions. Soon there will be far more pieces of DNA that were synthesized starting with one primer thus has a definite stopping point (the DNA ends) when the synthesis starts with the other primer and goes the other way. When that happens the DNA will have a definite stopping point on both ends and all new DNA made will be of a fixed length. A picture shows it best. I have put in some (short) primer regions.

STARTING SITUATION (after DNA is denatured)

<--------------------GTAGCAGT------------------------------CCTAGGTA-------------------------->
(starts here) CATCGTCA------------------------------GGATCCAT-------------------------->

<--------------------CATCGTGA------------------------------GGATCCAT (starts here)
<--------------------GTAGCACT------------------------------CCTAGGTA-------------------------->

NEXT ROUND OF AMPLIFICATION

CATCGTCA------------------------------GGATCCAT-------------------------->
(stops here) GTAGCAGT------------------------------CCTAGGTA (starts here)

<--------------------CATCGTGA------------------------------GGATCCAT
(starts here) GTAGCACT------------------------------CCTAGGTA (stops here)

FURTHER ROUNDS OF AMPLIFICATION

CATCGTCA------------------------------GGATCCAT
(stops here) GTAGCAGT------------------------------CCTAGGTA (starts here)

CATCGTGA------------------------------GGATCCAT
(starts here) GTAGCACT------------------------------CCTAGGTA (stops here)

Thus at the end almost all of the PCR products with be only the short length. There will be a few longer pieces (one in each direction for each of the 40 or so cycles of amplification) but that will be such a small amount compared to the billions and billions of short pieces that you will not be able to notice them.

Let me know if you have any other questions.

Donna Hardy

Hi there,

Here is a website that has links to some really good PCR animations and explanations:

http://www.protocol-online.org/archive/posts/4025.html

Hope this helps!

Mary

Thanks everyone and especially for the links!!!

I completely forgot about the fact that the newly synthesised strand will actually have an end point due to the fact that it started with a primer! So once the polymerase reach this "end point" it can no longer synthesis anymore DNA!

When I was trying to work out the PCR process in my head I just took the "primer" as part of the normal DNA template ie. I forgot that once you pass the primer, there are no more nucleotides!

Again, thanks everyone!!!!!!!!!!!