Obtaining cDNA 5' sequence

Casey Finnerty cmf5 at cornell.edu
Thu Dec 16 17:53:41 EST 1993


Netters:
After screening a lambda ZAP II expression library with polyclonal serum,
I obtained several positive clones that I subsequently sequenced. On
Western blot, the size of the expressed protein is close to the size of
the protein used to prepare the antiserum (app. 33-35 kD). Unfortunately,
after sequenceing, it looks like I only have a reading frame accounting
for about 29 kD of the protein. The first ATG occurs too far downstream
to be the start codon, so it looks like these clones are missing the 5'
end. I understand this to be a common problem of cDNA library
construction that results when the reverse transcriptase drops off too
early.
	In order to obtain the 5' sequence, I've come up with three approaches
and would appreciate your comments/suggestions.
	1) Rescreen the cDNA library using a DNA probe prepared from one of the
nearly full size clones on hand. I'm trying this currently, but so far
have only pulled out one clone that I have not yet sequenced.
	2) Use a 5' fragment of the largest cDNA clone to sequence directly off
transcripts extracted from tissue. The protein is fairly abundant when
induced (app. 1%); should there be enough transcript to prepare cDNA for
sequencing?
	3) Use PCR-based RACE or SLIC to amplify the 5' end from either the cDNA
library or transcripts. I've gotten varying estimates of the success rate
of these methods. That fact, combined with the price of the kits (app.
$400 from Gibco/BRL or Clontech) urges me to solicit advice. Do these
approaches work? Is there a cheaper way to do this by buying individual
enzymes and reagents?
	Your comments on these approaches and suggestions for any others are
sincerely appreciated.

Casey M. Finnerty
Boyce Thompson Institute at Cornell University
cmf5 at cornell.edu
607-254-1262



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