Rep (2) : a good PCR mimic?

Richard Pelletier rpelle1 at po-box.mcgill.ca
Thu Jun 27 12:57:36 EST 1996


In article <4qte1v$orc at mserv1.dl.ac.uk>, Mr SHIRE David
<David.SHIRE at TLS1.elfsanofi.fr> (Tel 61.39.96.00) wrote:

> > In article <31CABDFE.58D9 at uia.ua.ac.be>, bgrobben at uia.ua.ac.be (Bert
> > Grobben) wrote:
> > 
> > > Hello,
> > > 
> > > I'm currently trying to optimize a quantitative RT pcr for a certain 
> > > mRNA in different rat tissues..........snip
> 
> > 
> > The extra PCR product you got is probably a recombination product. Indeed,
> > when performing PCR with 2 templates of similar sequence, there is a
> > polymerase template switching that can occur (it has been documented in
> > the litterature) and create extra bands. If you are performing competitive
> > PCR, this is no concern since these extra bands compete for the template
> > and the competitor (mimic) equally, at least in theory. Your technique for
> > mimic construction seems very complicated and time consuming. You can
> > synthesize a competitor by low temperature primer annealing, using your
> > primers and your cDNA as template. As you will get many bands, you select
> > by choosing a band that is shorter that your regular product, but
> > necessarily contains the same primer binding sites. This technique and
> > many others have been described in Biotechniques in the last 1 or 2 years.
> > Hope this is helpful. Also look in Nucl. Acids Res. for similar protocols.
> > 
> > -- 
> > Richard Pelletier
> > McGill Experimental Medicine
> > 
> > 
> A criticism of this method is that, although the primers amplify these
secondary sequences they don't necessarily match the priming regions 100%,
because they are low stringency primers. Consequently there will be a
difference in priming efficacy between the target sequence and the
'control' sequence, therefore useless for quantitation.
> 
> David

Mr. SHIRE, 
         you did not understand the principle of the method. It consists
in using the same primers as for the target, but with a lower annealing
temperature. This will cause the primers to anneal to non specific sites,
therefore amplifying regions with no sequence homology with the target
product. However, since the technique uses the same primers, the sequences
at the ends MUST BE 100% homologous to the primers: the first PCR cycle
generates a molecule with one primer at the end and a non specific
extremity at the other end. The second cycle generates a molecule that
contains both primer binding sequences at the ends, and so on for the
subsequent cycles. I used this method successfully to perform competitive
PCR (with high stringency annealing), and there is no difference in
priming efficacy between the target and the control sequence, since BOTH
have exactly the same ends, although the internal sequences differ. The
reference to this method is: Forster, E. (1994) Biotechniques 16:
1006-1008.

-- 
Richard Pelletier
Experimental Medicine
McGill University



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