how to distinguish a doubled codon
pjie2 at cam.ac.uk
Fri Sep 9 02:43:41 EST 2005
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
>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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