how to distinguish a doubled codon

[Han]

Thanks, Peter!

(for brevity topposted, against my rules <grin>

Han

Peter Ellis <pjie2 at cam.ac.uk> wrote in
news:MPG.1d8b4d8b7aaae8d5989cb3 at news.individual.net: 

> 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
> 



-- 
Best regards
Han 
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