DNA degredation during PCR?

R. Burkert burkert at biotec.uni-bremen.de
Mon Feb 21 15:59:55 EST 2000


When I understood you right, you have a problem with your nested PCR-
I have worked quit a few times with different nested systems, and there are
many problems which could be the reason for getting no result.
So if you don't mind, I'll ask a few quexstion....


> In the first reaction, the bulk DNA is
> denatured and primers anneal and then continually amplify your region of
> interest so by the end of the reaction, your sample is highly
> concentrated with amplified product.

What kind of material do you use? Is it fresh, and when, why do you think it
is
nessesary to use a nested PCR? If it is fixed material, how do you prepare
it?

> My question is, what happens to
> the rest of the unprimed, unamplified DNA template?

Usually the amount of original DNA is compared to the number of produced
copies
irrelevant- long fragments of cellular DNA will surely break into smaller
pieces, but
this has no effect for the reaction.

> Does it degrade
> during the PCR cycling due to the temperature range (94C - 45C)?  If so,
> why doesn't the product degrade as well?  Or does it as well, leaving
> you with product that is less than the potential because of degradation?
>
> I ask this because I got no amplification of my nirS gene in the first
> PCR ran, used reaction of that as template for a nested PCR ran with a
> different forward and reverse primer and got no product.  I would have
> thought that some of the original bulk DNA that went into the first
> reaction may still be present in that 2 ul that I used as template for
> the nested reaction.  If this was so, I could have product after the
> nested (2nd) PCR reaction with the different primers amplifying the bulk
> DNA.

I don't think that you could get enough of the original DNA in your second
reaction, because you should only use a small amount from the first reaction

to transfer it to the second reaction-
For example, if you do a PCR with 50 µl you shouldn't take more than 0,5-1
µl
as templat for the second reaction, first the number of copies would be too
large
(amplification wouldn't work at all), second the amount of salts from the
first
reaction would influence the second if you transfer a larger amount.

So how Reaction volume did you use as template for the nested reaction, and
how many cicles did you run?





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