PCR mutagenesis?

daniel h schott dhs3 at cornell.edu
Mon Apr 17 11:00:37 EST 1995


In article
<Pine.SGI.3.91-heb-2.05.950416203628.13500A-100000 at ccsg.tau.ac.il>,
spira at CCSG.TAU.AC.IL (spira beny) wrote:

> I want to make a PCR-random-mutagenesis by adding a smaller concentration 
> of one of the nucleotides. Does anyone has a protocol or a reference for 
> such a method? Thanks in advance,
>                                  Beny Spira
> Beny Spira
> Dept. Biochemistry
> Tel-Aviv University  Israel
> E-mail: spira at ccsg.tau.ac.il

Here's a few references that might help:

Eckert, K.A. and T. A. Kunkel. (1990) High fidelity DNA synthesis by
Thermus aquaticus DNA polymerase. Nuc. Acids Res. 18:3739- 3744.

Leung, D.W., E. Chen and D. V. Goeddel. (1990) A method for random
mutagenesis of a defined DNA fragment using modified polymerase chain
reaction. Technique (A. J. Cell. Mol. Biol.) 1:11- 15

Muhlrad, D. and R. Parker. (1992) A rapid method for localized mutagenesis
of yeast genes. Yeast. 8:79- 82

Cadwell, R. C. and G. F. Joyce (1992) Randomization of genes by PCR
mutagenesis. PCR Methods and Applications 2:28- 33 (Cold Spring Harbor
Laboratory)


For many people's purposes, standard PCR using Taq polymerase and [Mg] >>
[dNTPs] gives a more than high enough mutation rate. If you need an
extremely high mutation rate, try the following conditions from the last
reference:

0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,1 mM dTTP, 7 mM MgCl2, 0.5 mM MnCl2
(make fresh and add just prior to enzyme), and 5 u/100 ul Taq polymerase.

I hope this helps,
				-Daniel Schott

Daniel Schott
E- mail: dhs3 at postoffice.mail.cornell.edu



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