It is often necessary to make multiple amino acid substitutions at a particular site to determine the function of the wild-type amino acid in protein structure and function studies. Each substitution requires a unique mutation at that site. An alternative to making a series of predetermined substitutions is to create a library of mutations at that site that encompasses all possible amino acid substitutions. The creation of such libraries is often simple. Screening the number of clones necessary to insure a complete representation of substitutions can be difficult and time consuming. Amino acid substitution by amber suppression provides an alternative to standard site-directed mutagenesis and library approaches (1 ).
