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hybrid5228 08-30-2010 05:09 PM

Plasmid PCR question...
I am a biochemist working with a bunch of physicists, and an interesting point came up the other day while discussing PCR in plasmids.

At the end of the extension, you are left with a nicked piece of DNA. The nick appears between the beginning of the primer, and the last nucleotide added to the circle. During the next cycle, heating the dsDNA to 95 degrees melts the two strands apart. Why does the nicked strand not linearize? How is it that you end up with circular product?

The next round of PCR should start by adding a primer and extending the chain over the nick to form circular product. How is the polymerase able to reconcile this nick, possibly linear at this point? How does it find where to start replicating the chain?

I would never have thought about this without them asking me, and tech consultants from Stratagene have been unable to answer this question... I am hoping someone on here can lend insight.

Is it due to DNA persistence length? Stacking energies? I have no idea.

kevin lee 08-31-2010 05:06 AM

Re: Plasmid PCR question...
Hope you can have a better idea then!

superbeamer 11-17-2010 03:18 PM

Re: Plasmid PCR question...
I also have the same question (I'm the physicist working with biochemists!). I'm trying to obtain linear ssDNA and was thinking about doing PCR on a single-stranded plasmid with only one primer. After many cycles, would this give me mostly circular DNA or linear?

brave_beaker 04-14-2011 10:21 AM

Re: Plasmid PCR question...
Hope all of you have a better idea soon :)

4N6addict 04-14-2011 10:02 PM

Re: Plasmid PCR question...
Asking why you would end up with a circle is like asking why the primer adheres to your template in the first place. You have all these fragments moving around, and when one of the "nicked" ends in the circle adheres to one side of the strand you are trying to insert, at that moment, you do have a linear piece. But why did they adhere in the first place? Because they are complementary. Similarly, the other end of your circular DNA complements the other end of the insert. So they are likely to combine, in fact, they would prefer it (if given the choice). Their close proximity enhances the likelihood for recombination even more. This is why you have to look for dimers, etc, when choosing a primer, because you don't want them to adhere to themselves!

I don't have any hard evidence, but I would say far and away the majority of your amplicons are going to be circular. I really can't imagine ending up with a lot of linear strands.

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