Mutagenesis -- One primer or two?

Yi, Xiaoming Z3C20 at ttacs3.ttu.edu
Thu Apr 13 19:05:47 EST 1995


In <3mjfo2$846 at news.ycc.yale.edu> Gary writes:

> 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

There are actually some methods based on this idea. I can give you two old
ref.:Hofer, et al., Gene(AMST), Vol. 84, 153-157,(1989); Slilaty, et al, Anal.
Biochem., Vol. 185, 194-200,(1990). I saw one in the recent issue of
BioTechniques too. Good luck.

xiaoming Yi
Texas Tech University



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