How "random" are random primers?

Richard T. Pon rtpon at acs.ucalgary.ca
Thu Mar 9 11:10:40 EST 1995


In article <1995Mar9.223714.1 at cc.newcastle.edu.au>,
 <mdcabl at cc.newcastle.edu.au> wrote:
>Hi all,
>
>A friend of mine asked an interesting question.  What is the G-C content of a
>random hexamer?  Before you flame, I know that it would be 50% if it is 
>truly random sequence.  The more relevant question is perhaps is a random
>hexamer truly random or is the G-C content decreased to increase the annealing
>temperature of the hexamer.  My friend had no luck with the local tech-reps. 
>Can someone out there help my friend out?
>
>Cheers,
>Allen Black
>Dept. of Pathology
>Univ. of Newcastle
>mdcabl at cc.newcastle.edu.au
>
	The composition of a "random" hexamer, or any synthetic
oligo containing mixed base positions will depend upon how it was
made. A long time ago, we investigated how accurate the on-line
base mixing was on our automated DNA synthesizer. We made
dinucleotides and analyzed the products by HPLC. We found
significant deviations from the expected ratios (after taking the
different extinction coefficients into consideration).  This was
attributed to a mechanical effect (not because of an inherent
difference in the reactivity off the different bases. Although,
we only tested a couple of instrumennts, I have no reason to
believe that the effect doesn't occur with any other synthesizer
which makes degenerate base additions via simultaneous opening of
multiple valves.
	To compensate for this effect, our facility makes randdom
hexamer and any other sequence which requires critical control of
degeneracies by manually preparing a mixed base solution and
using only a single base port in the synthesis.

Richard T. Pon
Director, UCDNA Services
University of Calgary
4403-220-4277, FAX: 403-283-4907
 




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