Primers with dI vs mixed bases?

Allen Gathman C741SCB at SEMOVM.SEMO.EDU
Tue Aug 30 18:54:24 EST 1994


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.

Allen Gathman  C741SCB at SEMOVM or C741SCB at SEMOVM.SEMO.EDU
Biology Department, Southeast Missouri State University



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