Mutagenesis -- One primer or two?

Gary Rudnick gary.rudnick at
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

Gary Rudnick

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