Point Mutation Problem

Mike Perry mike.perry at bris.ac.uk
Mon Jul 7 05:29:40 EST 1997


Kelly R. Pitts wrote:
> 
> I am currently working on developing a mutant protein.  Using a PCR based
> technique with my gene of interest in a pUC vector, I successfully changed
> the codon AAG to GCG (K to A mutation by changing the first two
> nucleotides AA to GC), as evidenced by sequencing.  I isolated the mutant
> insert from pUC and cloned into pcDNA3.  Sequence confirmed the presence
> of the mutation.  I then isolated mutant insert from pcDNA3 and cloned
> into pEGFP-C1.  Sequence showed that the mutation reverted back to the
> wild type nucleotides!  Several attempts always showed a reversion of
> mutation.  Thinking that there could be some contamination, after
> isolating the mutant gene from pcDNA3, I sequenced both the pcDNA3/mutant
> construct AND the gel isolated mutant insert.  BOTH showed the presence of
> the mutation.  I did the ligations (mutant insert into pEGFP-C1), isolated
> clones, and sequenced.  Sequence showed a reversion of mutation.
>    Does anybody have any ideas?  Could it simply be a contamination
> problem?  My concern is that the mutant insert sequenced positive for the
> mutationÑÑÑwhat else can go wrong between the ligation and transformation
> (except contamination?)
> 
> Kelly Ray Pitts
> Mayo Clinic
> pitts.kelly at mayo.edu

Could the mutation be lethal, so that a small amount of contamination
with native sequence comes through? If there's a low level
contamination, it may not show up in the sequencing. Although why the
mutation should be lethal in pEGFP-C1 and not pcDNA3 is anybodies
guess!!!
-- 
*************************************************************
Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK
BS8 1TD
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