Point Mutation Problem

Taek H. You tyou at postbox.acs.ohio-state.edu
Thu Jul 3 04:12:24 EST 1997

In article <pitts.kelly-0207971150110001 at reeves.mayo.edu> pitts.kelly at mayo.edu (Kelly R. Pitts) writes:

>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?)

I don't know this is proper answer to solve your problem.
But, if your product is hemizygous (i.e. one strand is mutated, the other 
strand is original sequence), bacteria will correct the mutation with their 
own repair system.
I think generally, majority of the PCR product would be the mutated strands. 
If that's the case, I have no clue.

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