Procedure for producing a collection of mutants of a coding sequence inserted in a plasmid by identification or selection of restriction sites

摘要

Producing a collection of mutants of a coding sequence inserted in a plasmid, is new. The procedure consists of the following stages: a) identifying or selecting one or more restriction sites not contained in the coding sequence and the plasmid; b) preparing an intermediate mutant in parallel for each codon to be mutated in the coding sequence, in which either the codon to be mutated is replaced by a sequence containing one of the identified restriction sites which is inserted in the codon or at the level of 10 nucleotides on either side of it; c) effecting a mutation from each intermediate mutant that allows a replacement of the sequence containing the restriction site by each of the codons corresponding to 19 amino-acids not present in the initial coding sequence or a sub-assembly of them while restoring the framework of the initial reading, and d) digesting the product resulting from stage c) by the specific restriction enzyme of the restriction site inserted in stage b).