Kunkel mutagenesis

s.a. anyone at medcn.umontreal.ca
Tue Sep 9 18:49:05 EST 1997


Harry Witchel <Harry.Witchel at Bristol.ac.Uk> wrote in article
<341423A8.6E1A at Bristol.ac.Uk>...
> Hello Molecular Masters --
> 	Can anyone describe to me the elements of Kunkel style 
> mutagenesis?  I am told it is an alternative to large scale PCR 
> mutagenesis, which gives me a lot more trouble than either small scale 
> PCR mutagenesis or long range PCR.  My gene is over 3 kb and 65% G-C.
> 	Many thanks,
> 	Harry
> 

Hello witchel, 
I am bored to do kunkel mutagenesis. The idea is to produce single stranded
(ss) template from your plasmid or RF. Usually ss is produced in a E.coli
host deficient in synthesis/processing of Uracyl ( a Unc-dug- strain like
E.coli CJ236 the most comun used). The ss is extracted from the cells,
annealed with your mutagenic oligo carrying at least a 5' and 3' arms of
20nt or so complementary to your ss. I have been able to mutate even 20 nt
or more with the same oligo, just it requires very big oligos (remeber you
have to include the complementary "arms"). after annealing of your oligo
you do a fill-in reaction using T7 polymerase or klenow (T7 works better)
and you transform the fill-in product in a normal E.coli strain. The idea
tu use CJ236 as a host to produce ss is because the efficiency of getting
the double mutant in both strands after fill-in increases by 80% or so.
Remember that theorically you have 50% to get the mutation in your ds! So
the mutant strain producing a Uracyl-containing ss makes the frequency of
your mutant be bigger than your w.t (more details, see a methods enzymology
of Kunkel, 1990). So the problem of Kunkel is the following: 
1. You need to have a vector than you can produce ss (like a phagemid,
phage vector, etc.)
2. You need to identify the clones carrying the mutations by restriction
analysis (if the mutagenesis introduces a restriction site), or by
sequencing.

But it works wanderful!

Hope this will help you witchel!
Good luck, 
Pilar
Oups! my adress is Blancafp at magellan.umontreal.ca




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