hot mutagenesis techniques

brett brett at BORCIM.WUSTL.EDU
Tue Nov 21 11:40:37 EST 1995


>Hi all,
>I've recently cloned a protein that binds (with differing affinities) a family
>of structurally related ligands. I have the sequence in pMalC2 (expression)
>and pGemT (original
>cloning from PCR). I'd like to subject the sequence to random discrete
>mutagenesis in order to obtain a family of related proteins that possess
>altered  relative
>specificities and affinities for the family of ligands. 
>Is there one or mure easily executed mutagenesis techniques that people might
>recommend? I have a decent screening assay.
>
>Thanks in advance for any help.
>
>Cal Vary

Try PCR under reduced stringency: NAR v21 p777; PCR Meth & Appl v2 p28; Anal
Bioch v224 p347; Technique v1 p11; Biotechniques v11 p204.
Also of interest might be DNA shuffling: PNAS v91 p10747.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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