Oligonucleotide-directed mutagenesis

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

In the Ca-ATPase from sarcoplasmic reticulum, oligonucleotide-directed, site-specific mutagenesis has been applied to identify amino acids involved in Ca binding. Mutation of 30 glutamate and aspartate residues, singly or in groups, in a stalk sector near the transmembrane domain has little effect on Ca " -transport. In contrast mutations to Glu ° , Glu, Asn , Thr , Asp ° or Glu ° resulted in loss... 

Zoller, M. J., and Smith, M., Oligonucleotide-Directed Mutagenesis Using M13-Derived Vectors an Efficient and General Procedure for the Production of Point Mutations in Any Fragment of DNA. Nucleic Acids Res., 1982. 10(20) pp. 6487-6500. 

Cloning of constructs was performed according to standard protocols [Sambrook et al. 1989], The mutations were created in the plasmid pBluescript II KS (+) by oligonucleotide-directed mutagenesis. 

Michael Smith and Kary B. Mullis Chemistry Oligonucleotide-directed mutagenesis and polymerase chain reaction... 

DNA synthesis, primed in part by the oligodeoxynucleotide, leads to heteroduplex plasmid circles. (Source After G. Dalbadie-McFarland, L. W. Cohen, A. D. Riggs, C. Morin, K. Itakura, and J. H. Richards, Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function, Proc. Natl. Acad. Sci. USA 79 6408-6412, 1982.)... 

Oligonucleotide-directed site-specific mutagenesis is a powerful technique for the study of proteins and has brought about great progress in research on the structure and function of proteins. This technique makes it possible in theory to obtain any mutant enzyme desired. One of the goals of such protein engineering is to make the proteins more stable. 

Merino E, Osuna F, Bolivar F, Soberon X, A general PCR-based method for single or combinatorial oligonucleotide-directed mutagenesis on pUC-M13 vectors, Bio techniques, 12 508-510, 1992. 

Figure 6.36. Oligonucleotide-Directed Mutagenesis. A primer containing a mismatched nucleotide is used to produce a desired change in the DNA sequence.

Several point mutations in the S. cerevisiae flavocytochrome coding sequence have been constructed by oligonucleotide-directed site-specific mutagenesis of the cloned gene (143). The enzyme-coding region was transferred to a plasmid designed both for expression of... 

Substitutions Oligonucleotide Directed Mutagenesis. Mutant proteins... 

Sidhu, S. S. and Weiss, G. A. (2004) Constructing phage display libraries by oligonucleotide-directed mutagenesis, in Phage Display A Practical Approach (Lowman, H. B. and Clackson, T., eds.), Oxford University Press, Oxford, UK, pp. 27-41... 

---