Taq and dITP

Basavaraju Shankarappa bsh at MED.PITT.EDU
Wed Apr 20 08:37:15 EST 1994


> Dear netters,
> just a simple question. I want to introduce random mutations in a short
> DNA sequence by PCR using  dITP instead of dGTP. Does Taq (or Vent) accept
> dITP (e.g. T7 does) ??
> Any suggestions
> Thank you
> Robert
> -- 
> * Robert Slany Ph.D., Institut fuer Biochemie, Fahrstr. 17, Germany *
> *    Tel. 0049(GER)-9131-854196, Fax: 0049(GER)-9131-854605         *
> *       mfb102 at cd4680fs.rrze.uni-erlangen.de (Internet)             *
> *              ----------Don`t panic-----------                     *

	Yes.  Spee et al have a publication in NAR (21:777) that 
describes this method of mutagenizing a given sequence of DNA.  The
bottom line is with 14uM of one selected dNTP + 200 uM of dITP.  Do
this with four reactions, pool them and clone them out.  They observed
mutations of 37.5 per 10-4 using this protocol.  
   
Raj Shankarappa 
bsh at med.pitt.edu
745 Scaife Hall
Department of Pathology
University of Pittsburgh School of Medicine
Pittsburgh PA 15261
Tel: (412) 648-9763 
Fax: (412) 648-1916
Home: (412) 823-3504




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