Synthesis of oligos with a 3' N
Ransom Hill Bioscience
visla at ix.netcom.com
Fri Sep 26 10:02:43 EST 1997
One additional note. The loadings on the various CPGs vary wildly. You can purchase small quantities of CPG in bulk, i.e., not in columns from your supplier and these will list the "micromole/gram" loading. Then you can use these to bias the mixture of "N" CPG you prepare.
It is not uncommon to find an "A" CPG, for example loaded at 15 micromoles/gram and a "G" loaded at 35. In such a case you would be best served by mixing a 2:1 ratio of A to G.
For the remainder of the bases, what we do is actually mix the amidites and use the "alien base" port. This is best for two reasons: (1) No synthesizer can mix as well as you can, (2) the kinetics of incorporation vary, due to steric effects that not only derive from the difference between pyrimidine and purine, but among the protecting groups. T, for example requires no protecting group (no primary amine), is a pyrimidine, and is therefore incorporated at a rate slightly higher than expected. A and C are the slowest (bulky benzoyl protecting groups). We bias our amidite mixtures when we do this, and as a rule of thumb you could very closely approximate the empirically derived ratios by increasing A by 2%, C by 3%, and decreasing T by 5%. G will be the remainder.
I hope this helps, and if not, maybe it was interesting.
Michael MacDonell, Ph.D.
In article <+3gXsMAFB3K0Ewtq at demon.co.uk>,
"Dr. Duncan Clark" <duncan at genesys.demon.co.uk> wrote:
>What's the easiest or most reliable way to synthesise in house an oligo
>with a 3' N - combine four columns, A C G & T, in one or synthesise 4
>separate oligos and combine. What's the general approach for
>synthesising random hexamers or nonomers?
>The problem with being on the cutting edge is that you occasionally get
>sliced from time to time....
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