Dear Gregory and Darren,
Thank you for your answers. They have been very useful.
it possible that the protein is toxic (even when slightly expressed from your possibly leaky pET vector), so that e.coli select for mutations that kill expression of your recombinant gene ...
2012/1/24 Darren Hart <[log in to unmask]>:
> I think the explanation is this:
> The source is natural viral RNA which is a mixture of naturally mutated
> sequences (e.g. flu forms such a quasispecies)
> See:
> http://www.virology.ws/2009/05/11/the-quasispecies-concept/
>
> The pooled RNA has an average sequence that you see when you sequence the
> pooled cDNA (individual mutations are hidden by the averaging effect of
> having many sequences present).
>
> But when you clonally separate DNA molecules by transformation (1 plasmid
> enters 1 cell to yield 1 colony), you see each individual molecule
> represented 100% in the sequencing chromatogram from the plasmid DNA that
> you have isolated from colonies.
>
> This is effect is commonly observed when sequencing influenza virus isolates
> from patients. It will have nothing to do with the E. coli strain. You can
> avoid it completely by using gene synthesis.
>
> Darren