one-step site-directed mutagenesis

Charles Brenner brenner at rose.brandeis.edu
Wed Apr 20 09:41:57 EST 1994


>Buddies,

>We would like to do some one-step gene disruptions using flanking regions 
>smaller than the recommended minimum.  Rothstein (Meth. Enzymol. vol. 194) 
>suggests using flanking regions of 100 bp or more on each side of the 
>selectable marker and the method we want to try gives us only about 50 bp on 
>each side.  Does anyone have information regarding homologous recombination 
>of fragments of this size?  Any input will be greatly appreciated.

>Dave Eide 

Dave,

I have gotten homologous recombination to work with a 153 bp PCR
product co-transformed with a linearized plasmid in an application
called One-Step Site-Directed Mutagenesis.

Here is a schematic diagram of DNA samples co-transformed:

       ====X============== 153 bp PCR product (mutations at X)
       24nt       <-40nt->
 //============== ============\\
 ||                           ||
 ||linearized 15 kbYEp plasmid||
 ||                           ||
 \\===========================//


To regenerate the plasmid, one needed homologous recombination on
either side of the site at which the plasmid was linearized (110 bp
and 40 bp).  However, to get site-directed mutants, recombination on
the left side had to be in the distal 24 nt.  

It turns out that linearized plasmids transform about as well as
closed circulars but addition of the PCR product boosted
transformation frequency by 3-fold.  The frequency of site-directed
mutants among the transformants was consistent with recombination at
an unbiased rate all the way to the ends of the PCR product.

Site-directed mutagenesis and ordinary gene disruptions should,
therefore, be possible with PCR-generated samples that have only tens
of nt of identity at each end.

The reference for One-Step Site-Directed Mutagenesis is
Brenner, Bevan & Fuller, Current Biology 3, pp. 498-506, 1993.

Best of luck,
Charlie Brenner

--
Charles Brenner, Ph.D.		Fellow of the Leukemia Society of America
Rosenstiel Center, rm 610	phone (617) 736-4944
Brandeis University		fax   (617) 736-2405
Waltham, MA 02254-9110		brenner at auriga.rose.brandeis.edu



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