a good PCR mimic?

Richard Pelletier rpelle1 at po-box.mcgill.ca
Wed Jun 26 17:31:33 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. In order to make a good quantification, I 
> needed a nice internal control. I first tried to create a deletion 
> mutant of the cDNA I'm working with, thereby reducing the length of the 
> PCR fragment from about 800 to 650 bp. 
> When doing PCR over 40 cycles on strong dilutions of plasmids containing 
> the full length cDNA, and shorter MIMIC cDNA, I always am confronted 
> with an intermediate PCR product, probably a heterodimer of a long and 
> short fragment. I tried to circumvent this by raising primer 
> concentration (I used 16 pmoles of each primer per 20ul reaction) and 
> lowering the plasmid concentration. But the intermediate stayed, while I 
> got a clear primer-band on my gel...
> 
> Probably it is better to synthesize another PCR-mimic, with different 
> sequence. I thought of doing this by annealing both primers to a 
> restriction digest fragment of a nice lenght (about 500 bp) with 5' 
> overhangs, by means of T4 RNA ligase (we have this available in the 
> lab), that anneals ssDNA or RNA. Afterwards, this product should be 
> blunted, cloned, and a selection must be made to select for fragments 
> containing different primers at the ends.
> 
> Has anyone experience with this technique, and does anybody knows 
> whether T4 RNA ligase can be used to ligate primers to blunt end 
> fragments also. I know there are more easy ways to create such a 
> fragment, by doing PCR with primers containing both 
> 'stuffer-DNA-primers' and primers for use in the quantitative PCR, but 
> that looks more expensive, since we have all the material for the T4 
> ligase method at our disposal...
> 
> PS: can anyone tell me how to subscribe to this mailing list (I suppose 
> this is a mailing list) since I wrote this article by WWW-ing to the 
> archives of this list on the net.bio.net-site....
> 
> Greetings,
> Stefan Nicolai
> Lab. Cellular Biochemistry
> Dept. Biochemistry
> University of Antwerp
> Belgium
> email: nicolai at uia.ua.ac.be

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



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