Reliable AND inexpensive! At the risk of starting a heated exchange, I must say
that often the least expensive service is not always the best.
However, more to the point, there should be many facilities which can prepare
satisfactory phosphorothioate oligonucleotides, including ours (e-mail to
dnalab@ucalgary.ca). If your colleague is concerned, he can ask specific questions
about which reagent is used for the sulphurization and how the sample was
desalted/purified. There are two common reagents for the sulphurization step, the
"Beaucage reagent" 3H-1,2-benzodithiol-3-one-1,1-dioxide and tetraethylthiuram
disulfide or TETD. In my opinion the Beaucage reagent is preferred since our
customers have found fewer non-specific toxicity problems with it. It is also the
reagent used for large scale antisense oligonucleotide synthesis for
pharmaceutical applications.
Purification of the phosphorothioate sequences is relatively simple since most
applications don't require high purity. However, it is important to remove
low-molecular weight impurities which can cause non-specific effects. In our lab,
we use a 40 cm Sephadex G-25 column to "desalt" our phosphorothioate products
after standard deprotection (NH4OH, 55o, 16 h). This is a much longer column and
(I believe) a more effective separation than obtained on the tiny NAP-1 columns we
use to desalt our other "phosphodiester" oligonucleotides. However, it doesn't
eliminate all of the N-1, N-2, etc. failure sequences, but has the advantage of
producing a large amount of material for the investigator to work with for a very
low price (i.e. no expensive HPLC separation required). Typically, we do 1
micromole scale syntheses and provide about 5 mg of desalted product. The
important point is to have a sample which is free of non-nucleotide impurities,
the presence of N-1, etc. failure sequences is not that important for most
purposes.
The amount of full-length product can be estimated by raising a typical coupling
yield (98-99.5%, should really be above 99%) to the power N, N= number of base
additions -1. If you colleagues oligo was quite long, than the 60% full-length
product might be typical. Usually, we recommend that sequences be kept to between
15-20 bases in length. Researchers should also always use a "control" sequence,
such as their sequence written backwards.
For more information on antisense oligonucleotides, a recent review by Richard
Hogrefe, "An antisense oligonucleotide primer" in Antisense Nucleic Acid Drug
Development 9, 351-357 (1999) is recommended.
Radhika Krishnan wrote:
> 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.
-- Richard T. Pon, Director, University Core DNA Services The University of Calgary, 3350 Hospital Drive N.W., Calgary, AB Canada, T2N 4N1 Tel: Lab. (403) 220-4277; Office (403) 220-4225. Fax: (403) 283-4907 Please direct DNA synthesis or sequencing orders to DNALAB@acs.ucalgary.ca
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