Method random diversification of a genetic sequence which preserves the identity of certain internal segments said genetic sequence

摘要

PCR method allowing randomly mutate a nucleotide sequence S, bounded at its 5 'and 3' by two segments F1 and F2, while preserving at the same time the identity of an internal segment D of said nucleotide sequence S, 5 characterized in that comprising the following steps: i) prone PCR is effected error throughout the sequence s using at least two primers, one of which is sense and the other antisense, respectively, F1 and F2 segments so that amplified the sequence S by introducing mutations randomly; ii) the amplification products obtained, which correspond to randomly mutated copies of purified S; iii) high-fidelity PCR is performed from the amplification products purified in step ii), using pairs of sense and antisense primers, respectively, at least: - a sense primer F1 and an antisense primer hybridizing in its all the D segment, to amplify the region F1-D copies of mutated S, - a sense primer anneals entirely with the D segment and an antisense primer F2, to amplify the region D- F2 copies of mutated S; iv) the amplification products obtained in step iii), consisting of copies of the F1-D and D-F2 sequence segments S mutated in step i) in which the D segment has retained its identity purified; v) a high fidelity PCR is performed from the amplification products purified in step iv) using antisense and sense primers, F1 and F2 respectively; vi) PCR products obtained, which correspond to nucleotide sequences S mutated in step i) in which the D segment has retained its identity purified.