neede: random mutations+doped oligo's

FJvanderWal vdwal at bio.vu.nl
Mon Jul 26 04:55:08 EST 1993


I want to change a sequence randomly by using doped oligo's  (N-N-C/G) in
which N stands for the wild-type nucleotides at the 1st or 2nd position of
a triplet, polluted with a certain amount of the other three nucleotides.
C/G stands for a mix of C and G at the 3rd position of a triplet; this is
to avoid the generation of the two stopcodons ending with a C or a G. The
part of the oligo in which I want a 'mutation' is 81 nt's (the oligo itself
will be a little longer to include some sites which can be digested after
annealing the 3'-ends and subsequent fill-in by a polymerase). Selection of
mutants is easy since we have a method in which upon transformation
wild-types won't grow on plates containing IPTG. My question is this:

How do I determine the frequency of polluted nucleotides, if I want to
synthesize a mixed batch of oligo's with only one mutation each?

Maybe you could provide me some references or formulas, any information
would be very helpfull!. Thanks in advance for your co-operation.

Fimme J. van der Wal

vdwal at bio.vu.nl



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