HELP please: Inverse PCR with genomic fragments!

gordon allison goa at aber.ac.uk
Fri Feb 9 05:47:50 EST 1996


In article <4fal3b$pd8 at sun168.rz.ruhr-uni-bochum.de>, 
user at garfield.neuro.ruhr-uni-bochum.de says...
>
>Dear netters,
>the following problem is driving me crazy: I try to circularize pools of 
>genomic DNA-restricition-nuclease-fragments of distinct sizes (2-4 kb). 
Among 
>these circular molecules there should be one that contains a certain gene 
>whose promoter region I want to clone. So far, I have tried to manage it 
by 
>inverse PCR with primers derived from the known protein-coding region. 
But 
>after 3x30 cycles there is no product! Another PCR-based method to catch 
>promoter regions is the Rapid amplification of genomic ends (RAGE). No 
>success, too. 
>Who has already solved the problems of a) effective circularization of 
>genomic DNA, b) suitable conditions for RAGE-ligations and RAGEs, and c) 
>inverse PCR with 0,001% target molecules among many others ?
>
>Thanks for any answer!
>
>Claus Graßmann, Department of Biochemistry, Ruhr-Uni Bochum
>
Dear Claus, 
	From what I have read, and also from my own work, the 
success of inverse PCR seems to depend upon the complexity of the genome 
from which you hope to amplify your product. I work with tissue from 
higher mammals and I have not managed to get up stream DNA sequences by 
this PCR technique, however, it does look brilliant on paper. Most of the 
success stories seem to be when samples from more simple organisms are 
used.

ps
 If anyoneout there knows how to tweek this method up, I would like to 
know.

			Regards, Gordon




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