ability to pcr introns from cDNA

Alexander Kraev kraev at bc.biol.ethz.ch
Mon Apr 29 09:01:45 EST 1996


>I was wondering if anyone has an opinion on the ability to pcr introns
from a cDNA prep...I understand that cDNA by definition is exon only, 
but
is it possible to isolate some pre-mRNA, and then reverse transcribe 
that
to cDNA, which could lead to pcr amplification of introns? We hope the
answer is no, but are unsure....thanks....<

First, if you synthesise your cDNA from total cellular RNA ( not 
polyA-selected), why shouldn't you be able to PCR introns? Second,
improperly spliced and "incompletely spliced" RNAs are rather often 
cloned in regular libraries, which were supposed to be made from polyA+ 
RNA. The presence of the latter in RT-PCR from total RNA is actually
even more frequent. The problem is, what is the steady state 
concentration of your unspliced message? You might opt for longer than 
usual primers and larger than usual number of cycles to catch it...


Alexander Kraev, PhD                     Internet:    
kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
> Swiss Federal Inst. Of Technology           FAX:      0041-1-632-12-13
> Universitaetsstr. 16                    Home Page: http://www.bc.biol.ethz.ch/BiochemistryIII/                                 
> CH-8092 Zurich                                     /Sasha/kraev.html





More information about the Methods mailing list