I am investigating various mutagenesis methods with the goal of
randomly introducing multiple changes into a cloned gene.
In particular, I am interested in the PCR (am I allowed to say PCR?)
based technique described by Leung, D.W., et al. (Technique 1:11-15)
and Cadwell, R.W., and Joyce, G.F. (PCR Methods and Applications 2:28-33).
I am interested in the use of nucleotide analogs in combination with
this technique. For example, DNA sequencing is often done with dITP
or 7-deaza-dGTP because the weaker base pairing interactions eliminate
certain gel artifacts. Since the PCR mutagenesis technique reduces
the fidelity of the Taq polymerase, these nucleotides might be
incorporated at a high rate. Perhaps this could be extended to other
nucleotide analogs. The major benefit would be a larger number of
mutations in fewer PCR cycles.
The ideal "universal" nucleotide analog will pair with any other
nucleotide and be randomly incorporated and not cause a large number
of frame shift mutations. However, I am willing to settle for
second best.
Does anyone know of any work regarding nucleotide analogs and base
pairing? Has anyone used analogs to investigate kinetics of various
DNA or RNA polymerases?
Larry Casson
lpcasson at phoenix.princeton.edu
