Mutagenesis using straight PCR?

Michael Benedik bchs1b at Elroy.UH.EDU
Mon Apr 20 16:03:27 EST 1992


In article <1992Apr18.200040.24096 at usenet.ins.cwru.edu>, txt15 at po.CWRU.Edu (Tao Tao) writes:
>
>To PCR specialists on the net,
>
>	I would like to know whether it is feasible to use standard PCR to
>random mutagenize a DNA fragment.  Since Taq polymerase has not editing 
>function, this should be possible.
>	My concern is the low rate (around 10^-4?), which means mutants would
>be hard to get, especially in the case of short target size.  So the question
>is this:
>	What parameters should I change in order to increase the error rate
>of Taq polymerase?  Also, is there any reagents useful in inreasing this error
>rate (base derivatives, etc)?
>	Thanks in advance for your help!
>
>Tao Tao

In addition to the Roufa reference mentioned above, there is a published
report of diretly using Taq for mutagenesis and getting reasonable 
mutagenesis rates. They report about 35% of the clones with mutations.
This makes it a simple and useful procedure but also raises serious
concerns about cloning with Taq and PCR.

reference is: Zhou, Zhang and Ebright. 1991. Nucleic Acids Research 
vol 19 No 21 pg 6052.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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