[Zbrafish] Re: Sequence homology for protein expression - help!

Christian.Broesamle from med.uni-muenchen.de via zbrafish%40net.bio.net (by Christian.Broesamle from med.uni-muenchen.de)
Thu Apr 24 02:29:55 EST 2008

Another possibility would be sequencing errors. Did you look at the
sequencing electropherogram (the colorful curves thing) to assess the
quality of your sequence that usually deteriorates towards the end of the
sequencing run and in particular at the positions in question? As to
whether the T to A substitution is affecting function, I think there is no
safe way to predict. There are some indications such as cross-species
conservation of the particular residue, location in a defined and
conserved functional domain or active site (phosphorylation site), that
would raise my concerns. But in the end, I don't think would want to take
the chances. What if your experiment doesn't work? You'd have to go back
and reclone and start all over  again. Is there a possibility you could
order an EST with the right sequence? You could PCR from this and likely
get the right sequence, because you start with A LOT of correct template
and need fewer cycles (or even clone by restriction digest).

> Hi Joanna,
> Thanks for the answer. The strange thing is that I do not get the same
> ‘errors’ when sequencing multiple clones. I can only think that this
> is due to the fact that the cDNA was made from multiple larvae
? Or
> that although I used HF the enzyme is causing errors. In any case,
> when I use blastx for protein sequence (part of the protein – 900bp) I
> get something like this:
>  Score =  446 bits (1147),  Expect = 1e-123
>  Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221
> (0%)
>  Frame = +2
> (One alanine substituting a threonine)
> Is such a substitution a problem?
> Thanks very much
> E.A.
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