how to distinguish a doubled codon

[Peter Ellis]

nobody at nospam.not wrote:
>Hi All:
>
>We have reason to believe that it makes a functional difference whether a 
>particular mRNA species has an extra codon or not.  Therefore we are 
>looking for ways to distinguish and quantitate whether (following PCR) we 
>have both a species with a single CAU codon in a certain place, or two 
>CAU's in tandem.  WE also believe that both transcripts may exist in a cell 
>at the same time, and would be interested in quantitating how much of each 
>is present.
>
>Can anyone suggest solution(s) for this problem?

1) Design a small amplicon covering the region of difference, do an 
   RT-PCR, run it out on a high resolution polyacrylamide gel and look 
   for the size change.

1a) As for (1), but clone the RT-PCR product.  Pick a large number of 
    clones and sequence them - see how many have one CAU codon and how 
    many have two.

2) Introducing the second CAU codon may well introduce or remove a 
   restriction enzyme site: check this out and see if you can get an 
   enzyme that cuts one isoform but not the other.  If so, design an 
   amplicon as in (1), do RT-PCR, digest with the appropriate enzyme 
   and run on a gel.  The advantage of this is that you're not forced 
   to look for a very small size shift.

3) Design two sets primers specific to the two different isoforms - 
   this should be trivially done by putting the extra codon at the 3'
   end of one of the primers.  Amplify each separately by RT-PCR and 
   quantify them separately.

Peter