neede: random mutations+doped oligo's
Richard T. Pon
rtpon at acs.ucalgary.ca
Thu Jul 29 12:39:05 EST 1993
In article <vdwal-260793105455 at mac133.bio.vu.nl> vdwal at bio.vu.nl (FJvanderWal) writes:
>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
See J. D. Hermes et al in Gene 84, 143-151 (1989). This article
is titled "A reliable method for random mutagenesis: the
generation of mutant libraries using spiked oligonucleotide
primers" and it contains a good description of the formulas
required to calculate "contamination" levels.
Richard T. Pon
Director, Regional DNA Synthesis Laboratory
University of Calgary
Phone 403-220-4277, FAX 403-283-4907 DNALAB at ACS.UCALGARY.CA
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