Building genes with oligos

Joe Mack mack at ncifcrf.gov
Sat Jul 16 15:30:54 EST 1994


>: Applied Biosystems 392 DNA/RNA synthesizer.  A typical
>: gene construction involves annealing 6-10 oligos,
>: ligation, cloning into a sequencing vector, and then
>: sequencing the products.  The problem is that the
>: resultant genes often carry one or more mutations.  These
>: mutations appear to be randomly distributed and are not
>: concentrated around the junctions between the oligos. 
>: The source of these mutations is not operator error or
>: errors in designing the oligos because in a bank of
>: clones coding for gene X, a given mutation will only
>: occur once while the rest of the clones are correct.
>: Transition, transversion and deletion mutations have been
>: observed.
>
>: Has anyone had any similar experiences?

I don't know if what has happened to you is unavoidable, but
it does appear to be common (see a paper in Gene about a year ago,
on the synthesis of a gene for HIV1 integrase, sorry forgot author and
don't have it with me here, the group was with Parke-Davis and when
I asked for the clone, they refused, unless we signed over all
rights to inventions that came of using the clone). They required two
rounds of mutagenesis to fix up the errors. So you seem to be doing much better.
	Joe Mack
	mack at ncifcrf.gov

>
>: Does anyone know what is causing the mutations?
>: Is it:
>:  a) Probabilistic errors in the oligo synthesis.
>:  b) UV exposure mutations (during UV shadowing).
>:  c) Secondary structure of the oligos.
>:  d) Recombination/repair
>
>: Thank you in advance
>: Glenn





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