I have a colleague who is looking for the literature reference from
Bio/Techniques between 1991-1994 about a paper in which the authors used
PCR and pfu polymerase to make site-directed mutations. It involved a
two step process. In the first step, the plasmid containing the cloned
fragment was used for a PCR reaction with one primer containing a
restriction site and the other primer contained the mutation to be
transfered. This PCR product was the first primer for the second PCR
reaction in which the second primer contained another restriction site.
The final PCR product was bounded by two restriction sites and contained
the internal transferred mutation.
If anyone has details of this method and a reference, it would be
appreciated.
