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-   -   Intrinsic mutation frequency in synthetic oligoes (http://www.molecularstation.com/forum/protocols-methods-forum/19135-intrinsic-mutation-frequency-synthetic-oligoes.html)

peter 03-12-2007 03:47 PM

Intrinsic mutation frequency in synthetic oligoes
 
Hi netters,
I need a method to assess the mutation frequency (purity) of
ologonucleotides - 50 -60 bp long. Any Idea how to do this?

Peter


newsnet customer 03-22-2007 09:30 AM

Intrinsic mutation frequency in synthetic oligoes
 
> Hi netters,

clone and sequence

St



Aawara Chowdhury 03-22-2007 12:01 PM

Intrinsic mutation frequency in synthetic oligoes
 
In <1173714423.081596.43410@c51g2000cwc.googlegroups. com>,
peter <[Only registered and activated users can see links. Click Here To Register...]> wrote:


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 36434455860060025978157675027927670979097959886449 930P | dc

peter 03-22-2007 02:38 PM

Intrinsic mutation frequency in synthetic oligoes
 
On Mar 22, 8:01 am, Aawara Chowdhury <[Only registered and activated users can see links. Click Here To Register...]> wrote:

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


DK 03-22-2007 09:44 PM

Intrinsic mutation frequency in synthetic oligoes
 
In article <seuMh.10918$[Only registered and activated users can see links. Click Here To Register...]>, [Only registered and activated users can see links. Click Here To Register...] wrote:

My non-expert understanding is that mass-spec is not particularly
(if at all) quantitative. If so, those spectra supplie by oligo
companies only detect gross impurities.

DK


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