Sequence-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... 

DNA sequence indicated that AMDase contains four cysteine residues located at 101, 148, 171 and 188 from amino terminal (Eig. 9). At least one of these four is estimated to play an essential role in the decarboxylation. The most effective way to determine which Cys is responsible to enzyme activity will be site-directed mutagenesis. To determine which amino acid should be introduced in place of active Cys, its role was estimated as illustrated in Eig. 13. One possibility is that... 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

The lactose transporter (lac permease) of E. coli is without doubt the most intensively studied and best understood of the bacterial proton-linked sugar transporters. Since its sequence was reported in 1980 [233] prodigious efforts have been made to elucidate its molecular mechanism by site-directed mutagenesis and other means. These studies have recently been reviewed elsewhere [234,235] and so will not be discussed in detail here. The important question for the present Chapter is whether the protein is related to the sugar-transporter family and so has lessons to teach us about their mechanisms. The permease is a 417-residue protein, and, like the other... 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

Wong, T.S., Tee, K.L., Hauer, B. and Schwaneberg, U. (2004) Sequence saturation mutagenesis (SeSaM) a novel method for directed evolution. Nucleic Acids Research, 32, e26. 

This company founded in 1994, is a contract molecular biology and screening service company for the pharmaceutical, research, and clinical markets worldwide. Custom services include gene synthesis, DNA sequencing, site-directed mutagenesis, nucleic acid purification, and genotype/genetic analysis. 

Changing the amino acid sequence of the cloned receptors by mutating nucleotides within the receptor cDNAs has proven to be an effective mechanism by which to identify structural features of the receptors responsible for their unique functional properties. In particular, site-directed mutagenesis has been employed to determine the ligand binding domains of each opiate receptor. 

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