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(NON) muatgenesis

Tony Meyn Tmeyn at ccit.arizona.edu
Mon Jul 25 16:15:42 EST 1994


In article <01HF4MDEHKIQ0001S3 at msvax.mssm.edu>, LAURETTE at MSVAX.MSSM.EDU
(LAURETTE) wrote:

> I'm trying to perform site-directed mutagenesis by Kunkel's
> method.  I am afraid of errors with PCR based methods, and having
> to sequence kB's of DNA.  There arwe not many unique sites that i
> can use to target a small DNA fragment.  The mutants are single or
> double amino acid substitutions OR deleting 100 amino acids (300
> nuc.).  I assume the DNA contains uracil since the transformation
> efficiency in CJ236 os 10^5 better than on MV1190. The single
> strand DNA seems to be correctly copyed, as i can see an shift
> upwards in the experimental (against the control) by DNA migration
> in an agarose gel.  I used either T4 (plus and minus gene 32 protein)
> and then thinking because I had 5.2 kB (insert + pGEX vector),
> used T7 DNA polymerase for the polymerization reaction. I also
> tried different bacterial strains, such as TG1, DH5a and W3110,
> instead of the original MV1190.  In all cases, using from 10-1000
> ng of DNA estimated from a gel , i got either no colonies or few colonies withou
> t any presence of the mutagenized DNA
> (the transformation is working properly with a control
> DNA). Is there someone who have experience with this kind of
> trouble and can try to help me?
> 

Laurette,

You are on a well travelled road.  I spent about a year there, before
taking the PCR offramp.  Some people have excellent results with the
Kunkel method.  I never got it to work for my particular mutagenesis.
It sounds as if you are trying the right things.  Especially important
is a clean ssDNA prep.  It sounds as if yours is OK.  Another problem
is non-specific priming, but as you are getting no transformants 
(as opposed to too many) this doesn't appear to be your problem
either.  

It could be that you are having problems with secondary structure.  
You may want to try higher temps.  I have also heard that using 
Klenow can help move the polymerization reaction past the secondary 
structure.  I've also read that Sequenase is useful in these
reactions.  

Another way to minimize some secondary structure problems would be
to sub-clone out the region of mutagenesis and use that construction.

I got my mutant on the first shot with PCR.  The insert on my construction
was only 400bp so sequencing the mutant was easy.  I would recommend
considering PCR if the Kunkel method continues to fail you.

Tony Meyn
University of Arizona



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