G + C rich sequences

nishir at ohsu.edu nishir at ohsu.edu
Fri Jul 23 15:07:13 EST 1993


I am trying to work with a 624bp cDNA sequence that is 74% G+C overall and
there is no smaller stretch that I can cut out that is lower than 70% G + C. 
We are having a terrible time with background when we try blotting with probes
made from this sequence.  If fact, even when we do PCR with exact match oligos
to this sequence we have difficulty amplifying one band (more than a smear of
others) from cDNA (even with more thermostable polymerases such as Vent and
going to melting temps of 95-97 deg). We would like to try some in situ
hybridization but are wary because of the background problems on blots.  I am
an amateur and do not understand fully the complexity of working with G + C
rich sequences.  Why is the background hyb (even to filter alone) so  high?
Should I try a different blocking agent when we prehyb? (we're susing salmon
sperm DNA). What's the main problem here?  Is it secondary structure?  Is there
alot of hybridization to repetitive DNA?  Does anyone out there have any
suggestions on how to deal with G + C rich sequences when doing in situs?

Rae Nishi
Cell Biolopgy & Anatomy
OHSU
Portland OR 



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