a good PCR mimic?

Bert Grobben bgrobben at uia.ua.ac.be
Fri Jun 21 10:46:57 EST 1996


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



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