HELP!!! E-coli (XL1-Blue et al) point-mutating my CpG island???
hmmoss at MAIL.MED.CORNELL.EDU
Mon Apr 14 16:22:34 EST 1997
I am in desperate need of a good detective to help me figure out a
-- I cloned 3 identical 19kb genes from a human genomic library (lambda
dashII expanded in XL1-Blue MRA P2) and subsequently subcloned fragments
into pBluescript (ranging from 260bp->4.8kb in size, and numerous overlapping
-- The e-coli strains I used for subcloning varied from plain XL-1
blue, XL1-blue MRF', to DH5alpha
-- Upon sequence analysis of my gc-rich promoter, I found that
several subclones had a C-->T subsitution within a CpG island. I know it is
NOT a sequencing problem because:
1) The region is in a high-quality sequence area
2) The presence of the 'C' confers a SacII site--> ergo, some of
mny clones cut with SacII, others do not!
-- I have obtained a consensus for the ENTIRE 19kb, and the ONLY
ambiguity lies in this region!!
THE BIG QUESTION: ?? Is it an effect of the seemingly identical 3
genomic clones -- does one clone have a non-essential point mutation??
OR is it an e-coli artifact????)
-- Oddly, the database/genome project has 2 fragments that match my
region: one was an EST and it contains the 'T', the other was a cosmid
clone that contained the 'C' --- this made me totally confused
MY QUESTIONS ARE:
1) What is the percent efficiency of these e-coli strains to
handle such CpG islands??? My assumption was that the INITIAL expansion
of the lambda clone in the XL-1 Blue MRA P2 would rid me of such a problem.
That is why I felt comfortable using more generic strains for further
expansion. (And even those strains have state-of-the-art genetic
modifications to handle CpG islands)
2) Could it be that this is a REALLY HOT SPOT??? Thus explaining
why even excellent strains have difficulty with it AND the fact that the
genome project is ambiguous?
(THE MUTATION IS RIGHT NEXT TO TANDEM STAT5 SITES IN THE PROMOTER,therefore,
it is very important to know the 'real' story)
Where did this mutation happen??? During the subcloning??? In one of the
original genomic clones?? I cannot figure out what is going on. Any imput is
appreciated. Unfortunately, I cannot trace which strain I used for which
clone (i do have a good notebook, but for most i just listed
"transformation" and not the strain ): ), and when I was subcloning I pooled
Thanks for your time!!!!!! Please respond personally via e-mail.
Heidi M. Moss
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