PCR of supercoils

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Fri Jul 27 10:55:57 EST 2001


In article <3B613BAD.50FF512A at Fackelmayer.de>, the eminent Frank O.
Fackelmayer at  wrote
>
>
>"Dr. Duncan Clark" wrote:
>> 
>> There was quite a long thread on the problem of PCRing supercoiled
>> plasmids in the 7700 listserv. Maybe it is worth checking the archives.
>> The basic argument, if I remember correctly, was that it should not be
>> possible due to the inability to 'unwind' the plasmid.
>
>in theory i would agree. Anyway, it works well in most cases. This does
>not say that the yield will be identical in all cases, but at least in
>the cases where we have directly compared the results with supercoiled
>vs. linearized plasmid we did not find a significant difference.
>or, to say it in other words: linearizing a plasmid has never helped us
>to get a PCR working that didn´t work with supercoiled plasmid.
>
>
>> It was argued
>> that the reason people can PCR off miniprepped DNA is that there is
>> sufficient nicked plasmid in the preps. 
>
>I don´t think so. We often successfully use CsCl purified DNA with no
>significant amount of nicked or linear plasmid as a template. Of course
>even such preps are never completely free of nicked plasmid, but if
>those little amounts were sufficient to allow for an amplification,
>noone should ever have problems in amplifying from "supercoiled" DNA, right?
>
>
>Frank

But you are not really comparing like for like if I follow correctly.

When you run a PCR out on a gel it is probably always after it has
reached plateau in the PCR so you wouldn't expect to see much in the way
of a difference in product yield even if you had a 10-100 fold copy
number difference at the start (obviously depending upon how many cycle
you are running but assuming that most people actually use too many).
With real time you will see the same rough yield at plateau but when you
are at quantification you are looking at when the PCR product is still
amplifying exponentially i.e. very early in the PCR. At that stage you
would see any differences in starting copy numbers that may be due to
differences in supercoiling.

I say may because I have yet to try this myself to see if there is a
difference.

It's worth a read of the 7700 archive.

Duncan  
-- 
The problem with the gene pool is that there is no lifeguard.

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382




More information about the Methods mailing list