Rep (2) : a good PCR mimic?
Mr SHIRE David
David.SHIRE at TLS1.elfsanofi.fr
Thu Jun 27 02:42:55 EST 1996
> 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.
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