Mutagenesis -- One primer or two?
gary.rudnick at yale.edu
Thu Apr 13 10:24:50 EST 1995
The "two primer" method of mutagenesis now sold as kits
by Stratagene, Clonetech, and probably others uses a selection
primer that removes a unique restriction site, and a mutagenic
primer for introducing the mutation of interest. It's supposed to
work on double stranded DNA, wheras the single primer (Kunkel)
method requires single-stranded DNA. My question is this:
Why couldn't one grow the plasmid in a strain that substitutes U's
instead of T's (ala Kunkel) and then mutagenize by transcribing in
vitro using a single mutagenic primer. Has anyone tried this? Is
there a reason that it wouldn't work. Is the ability to use dsDNA as
a template related to the use of two primers?
Thanks for any and all help
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