Omission mutagenesis

RVDESHP$ at PWINET.UPJ.COM RVDESHP$ at PWINET.UPJ.COM
Thu Aug 17 09:17:08 EST 1995


>would anyone have carried out a mutagenesis where a single residue was 
>omitted in the coding gene ?  would it be possible to use a primer with a 
>codon missing and generate the mutant by PCR amplification so that the 
>protein has a residue missing ?

Yes.  You certainly can design primer(s) with one or more codons missing.  
Depending on the number of codons you would like to omit, you would have to 
adjust the length of the oligo.  Typically, I would use a 30-33 base oligo 
to omit up to two codons.  Needless to mention, the omitted sequence should 
be at the center of the oligo.  In PCR, a 30-33 base oligo would have a 
high-enough melting temperature (65-74C depending on the G/C content) so 
that you can efficiently amplify your mutated sequence.

If you need to delete a larger portion of protein, say 5-10 amino acids, I 
would use "overlap PCR" with two mutagenic primers.

I hope this helps.  Good luck.

Rajendra V. Deshpande, Ph.D.
Molecular Biology Research
The Upjohn Company
Kalamazoo, MI 49007
e-mail : RVDESHP$@pwinet.upj.com



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