Site-directed mutagenesis

Name chandran at
Fri Feb 13 00:33:15 EST 1998

In article <886990729.1061355524 at>, bpaeng at wrote:

> Hi,
> I've recently identified modified enzyme activity of thymidine kinase in
> HSV-1. And found out that specific sequence is altered due to drug
> treatment.
> Now, I want to be more convinced of that site making drug resistance and
> would like make revertant using site directed mutagenesis.
> I pre-searched some major manufacturers of a vector having following
> necessities.  1. Must have a marker to identify mutation easily (eg.
> different antibiotics)  2. Must have regions to express either in
> prokaryote or eukaryote.
> But couldn't easily find appropriate one.
> Anyone recommend or know such one?
> Thx in advance.
> -------------------------------------------------------
> Brian J. Paeng
> Antiviral Screening Division.
> --------------------------------------------------------
> "I mutate everytime I illuminate, could I be reverted?" *phew*
> -------------------==== Posted via Deja News ====-----------------------
>     Search, Read, Post to Usenet

Try the QuikChange system from Stratagene.  Easy, fast, and highly
efficient (>90%!).  Basically, you make 2 complementary oligos containing
the mutation(s), then amplify with Pfu proofreading polymerase; digest w/
DpnI to get rid of parental DNA; concentrate the DNA, and transform. 
Screen colonies 
for mutations. It's working for me so well that I just sequence 2 clones
w/o even screening with a restriction enzyme.  Good luck! Kartik

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