Intrinsic mutation frequency in synthetic oligoes

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Thu Mar 22 09:38:38 EST 2007


On Mar 22, 8:01 am, Aawara Chowdhury <aaw... from FEMA-trailer.org> wrote:
> In <1173714423.081596.43... from c51g2000cwc.googlegroups.com>,
>
>  peter <peter.ianak... from gmail.com> wrote:
> > Hi netters,
> > I need a method to assess the mutation frequency (purity) of
> > ologonucleotides - 50 -60 bp long. Any Idea how to do this?
>
> Oligonucleotide purity is typically assessed by MALDI-TOF mass
> spec, or electrospray ionization mass spec (ESI).  There is a
> relatively recent paper, titled "High-throughput analysis of
> oligonucleotides using automated electrospray ionization mass
> spectrometry", by M.E. Hail et al. that compares the two
> techniques (Am. Biotech. Lab 22: 12-24).
>
> ESI was reliable for oligonucleotides upto 120 nt in length.
> MALDI-TOF was reliable up to about 50 nt.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc

Thanks,
ESI and MALDI is usually provided by the supplier. I want to know
minor (less than 1%)  fraction that might carry mutations. I was
thinking about sequencing, but that is quite brutal force and won't
give me results easily. Maybe I would make a construct with out of
frame AbtR and then I will clone my oligo (keeping AbtR out of frame)
and plate on antibiotic to see how many clones are resistant (e.g.
having oligoes that have 1bp deletions and repair the frame). Any
thoughts?
Peter



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