Primers with dI vs mixed bases?

John Watson ajwatson at romeo.caltech.edu
Wed Sep 7 11:22:55 EST 1994


In article <30AUG94.19339316.0033 at SEMOVM.SEMO.EDU>, Allen Gathman
<C741SCB at SEMOVM.SEMO.EDU> wrote:
> 
> I'm getting ready to order some oligos to use as primers for PCRing a
> protease gene from fungal genomic DNA.  The primers, to conserved
> catalytic regions in serine proteases, have been used by others to
> clone serine proteases from various organisms.  They are highly
> degenerate, largely because each one has 5 or 6 inosines in it.  What I
> want to know is, should I get primers made with inosines (which are
> relatively costly) or could I just have them made with equimolar mixture
> of A,G,and T at those sites?  The latter strategy reduces the effective
> concentration of the "right" primer, whichever it turns out to be; is
> this a major problem?  Would I have to compensate by increasing primer
> concentration?  Should I just cough up the bucks and get inosines?  I'd
> appreciate any information to help me make this decision.


If possible, bias your codon usage for the org in question -- you can often
eliminate 1 or 2 of the potential codons in your pool, obviating the need
for a fully degenerate location.  Obviously there's some risk in this --
the codon you eliminate, even if only used a small fraction of the time,
might just be the one in that location.  In cases where all four bases are
required, I always use inosine.  Unless I'm making probes for high
strngency applications (eg, in situ hybridization), in which case I use a
mixture of all four 'tides.  

Perhaps your facility can make very small amounts (for PCR I make 0.04
umol) to keep the cost down.

Good luck.

John
ajwatson at romeo.caltech.edu



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