how to distinguish a doubled codon
nobody at nospam.not
Fri Sep 9 06:00:56 EST 2005
(for brevity topposted, against my rules <grin>
Peter Ellis <pjie2 at cam.ac.uk> wrote in
news:MPG.1d8b4d8b7aaae8d5989cb3 at news.individual.net:
> nobody at nospam.not wrote:
>>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.
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