Simple question that is hard to answer

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Fri Feb 16 16:39:14 EST 2007


On Feb 16, 3:27 pm, "Peter Ellis" <p... from cam.ac.uk> wrote:
> peter wrote:
>
> > Duncan,
> > Your input is always appreciated, just my question was different and I
> > don't know how better to describe  - can one have two alleles of the
> > SAME Plasmid in the SAME cell, not different ori, not different
> > antibiotic resistances, everything is the same, just point muataion
> > that comes from cloning of heteroduplex DNA that has a mutation.
> > Peter
>
> It would seem unlikely.  Even assuming the heteroduplex molecule got cloned
> in the first place, my suspicion is that the first time it gets replicated,
> the mismatch would be "corrected" and you'd get two identical daughter
> copies of one allele or the other.
>
> Conversely, assuming you're transforming a mixed population of alleles in
> the first place, it's very unlikely that you'll get both alleles in the same
> bacterium - the whole *point* of the transformation is that you only get one
> plasmid into any given bacterium.
>
> You might end up with a heterogeneous population of plasmids inside the same
> bacterium, but that'll only happen if it's (a) a high copy number plasmid
> and (b) acquired the mutation within the bacterium.
>
> Peter

 Peter,
Mismatch will be corrected indeed, but using what template - it seems
that it will be corrected again 50/50 and still end up as mixed
population? If that is the case and I isolate DNA and re-transform, I
should be able to get 50/50 of homozygous plasmids in the cells and
have colonies that have one or the other allele. Right?
Peter



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