Point Mutation Problem

Kelly R. Pitts pitts.kelly at mayo.edu
Wed Jul 2 11:50:11 EST 1997

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

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