Promoter isolation

Bryan J. Maloney cavaggione at comcast.ten
Fri Apr 21 23:57:32 EST 2006


"Dr. Tribikram Bhattarai" <Tribikram.Bhattarai at uni-bayreuth.de> abagooba
zoink larblortch news:mailman.682.1145643385.16885.methods at net.bio.net: 

> I have isolated a stress related gene from cDNA library. The gene is
> active in all sorts of stress conditions. So I am interested on its
> promoter. I tried with inverse PCR to isolate the promoter but have no
> success. If you know any thing to get success by inverse PCR or if
> know other better methods to isolate a promoter please write me. 

This is pretty brute force and might not work for you.  Blast the human 
genome with your cDNA sequence.  This is likely to give you a hit on a 
chromosome.  Then you can find the BAC library clones that are predicted to 
cover the 5'-flanking region.  My group tries to get a clone that has not 
yet been published beyond the BAC insert ends.  Then, using the genome 
database sequence, we design PCR primers and pull a sequence out from the 
BAC.  We routinely get at least 2kb, although we usually shoot for 4kb.  
This is because we've found that there can be active elements this far away 
from the TSS, especially if we are looking for induceable elements.


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