Dear Radhika:
Short answers:
1) DNA providers? - The core DNA facility of the Dana Farber Cancer
Institute (Harvard Univ) lists most of the commercial DNA providers
(with hyperlinks to their corresponding Web sites), at
http://mbcf.dfci.harvard.edu/docs/DNAcomp.html
most of those companies post their prices at their Web sites, and
virtually all of them offer oligonucleotides with phosphorothioated
backbones.
2) Difficult to synthesize and purify? - Not that much.
3) Purification protocols? - This is better to be done by the DNA
provider, because they have more experience and will lose less time and
energy on that.
Nevertheless, general purification methods for synthetic DNA are
described at
http://www.interactiva.de/knowledge/nucleicchem/oligochem.html#RTFToC27
at the same site you will find other useful info such as what are the
phosphorothioates etc.
In addition, it is not a bad idea to have a good book on the thioated
oligos at hand, such as
"Applied Antisense Oligonucleotide Technology" (ISBN: 0471172790), 540
pp., 1998. The book is available from
www.barnsandnobbles.com for US$129.95.
(Disclaimer: there are other fine Web sites about DNA purification and
other fine books about antisense technology, I am not endorsing the
above examples, neither did I contribute to, or profit in any way from
them.)
Long answer:
I do not know details about your friend's complaint, but would like to
stress that sometimes it is not the DNA provider that provided a "bad"
product, but simply the DNA was ordered long and not purified.
I am not sure about the actual meaning of the "60% clean" product that
you mentioned:
A) The phosphorothioated backbones are synthesized by using a
sulfurizing reagent istead of regular iodine-based oxidizer. In
practical terms this means that during the addition of the monomer that
has to be with thioated backbone, the DNA synthesizer will use reagent
from the bottle "sulfurizing reagent" instead of the bottle "oxidizer".
The machine protocols are standardized and provided by the manufacturer
of the macine, the backbone is therefore "guaranteed" to be thioated, it
cannot be 40% phosphodiester and 60% phosphorothioated at the given
base. Nevertheless, some machines are better designed and their
protocols for phosphorothioated oligos more elaborated than others (I
would recommend the Expedite 8909, but others will probably disagree).
B) In view of the above, the 40% "not clean" probably means that only
60% of the final product was full-sized, and 40% were aborted products
and/or "mutants" (one base deletions, G-duplications etc.). This may
mean a bad synthesis, but it could also be a result of the length of the
oligo and the level of purity that your friend had ordered and the
provider guaranteed him.
For example, a synthesis of a longer oligo, a 50-mer, with 99% coupling
efficiency (what some DNA providers guarantee) will result in 0.99 at
the power of 49 full-size products, or 61%. The aborted products will
be about 40% (as was the case with your friend). The guaranteed
coupling efficiency could be lower with some other providers, a very
well known and big corporate provider of DNA guarantees 97% efficiency.
If the actual efficiency is close to 97%, even a "regular" 20-mer will
have 54% full-size products and 46% aborted products (for comparison,
99% coupling efficiency will give you 83% full-size product and 17%
aborted ones for the same 20-mer). The aborted products are not the
"fault" of the DNA provider, they will always be present in the crude
product due to the upfront limitations of the currently used DNA
synthesis protocols. If you want to remove most of the aborted
products, you can use one of the three purification methods: OPC
(oligonucleotide purification cartridge), HPLC or PAGE (listed in the
increasing order of price and complexity). There is also the question
of one-base deletions and other mutations, that cannot be fully resolved
with any currently available purification method, but these problems
should not be of concern to your friend, if he orders regular size
oligos.
Instead of simply changing the DNA provider, your friend should ask
beforehand the previous provider (or the new one) about their guaranteed
coupling efficiency (for the phosphorothoiated oligos!), the final
purity, and the price of the additional purification if he decides to
order one.
Best regards,
victor
www.alphadna.com
_____________________
Hi ABRFers,
This is a question posed by a colleague. He wants to know if there are
any
reliable ( and inexpensive) vendors who will synthesize oligos that
contain
sulfur backbone. The first vendor he bought it from did not provide a
very
clean product( only about 60% ). Are these difficult to synthesize and
purify?
Any purification protocols that he might follow?
Thanks in advance to any kind souls who know the answer.
Radhika.
City of Hope , Duarte, CA.
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