Method of PCR permitting random mutation of a nucleotide sequence S, delimited at its 5' and 3' ends by two segments F1 and F2, while preserving the identity of an inner segment D of said nucleotide sequence S, characterized in that it comprises the following steps: i) an error-prone PCR is performed on the whole of sequence S using at least two primers, one of which is sense and the other antisense respectively of segments F1 and F2, so as to amplify sequence S, introducing mutations into it randomly; ii) the amplification products obtained, which correspond to randomly mutated copies of S, are purified; iii) high-fidelity PCR is performed starting from the amplification products purified in step ii), using as sense and antisense primer pairs, respectively, at least: - a sense primer of F1 and an antisense primer hybridizing with the whole of segment D, in order to amplify region F1-D of the mutated copies of S, - a sense primer hybridizing with the whole of segment D and an antisense primer of F2, in order to amplify region D-F2 of the mutated copies of S, iv) the amplification products obtained in step iii), which consist of copies of segments F1-D and D-F2 of sequence S mutated in step i), in which segment D has preserved its identity, are purified; v) high-fidelity PCR is performed starting from the amplification products purified in step iv) using sense and antisense primers of respectively F1 and F2; vi) the PCR products obtained, which correspond to the nucleotide sequences S mutated in step i), in which segment D has preserved its identity, are purified. i) an error-prone PCR is performed on the whole of sequence S using at least two primers, one of which is sense and the other antisense respectively of segments F1 and F2, so as to amplify sequence S, introducing mutations into it randomly; ii) the amplification products obtained, which correspond to randomly mutated copies of S, are purified; iii) high-fidelity PCR is performed starting from the amplification products purified in step ii), using as sense and antisense primer pairs, respectively, at least: - a sense primer of F1 and an antisense primer hybridizing with the whole of segment D, in order to amplify region F1-D of the mutated copies of S, - a sense primer hybridizing with the whole of segment D and an antisense primer of F2, in order to amplify region D-F2 of the mutated copies of S, - a sense primer of F1 and an antisense primer hybridizing with the whole of segment D, in order to amplify region F1-D of the mutated copies of S, - a sense primer hybridizing with the whole of segment D and an antisense primer of F2, in order to amplify region D-F2 of the mutated copies of S, iv) the amplification products obtained in step iii), which consist of copies of segments F1-D and D-F2 of sequence S mutated in step i), in which segment D has preserved its identity, are purified; v) high-fidelity PCR is performed starting from the amplification products purified in step iv) using sense and antisense primers of respectively F1 and F2; vi) the PCR products obtained, which correspond to the nucleotide sequences S mutated in step i), in which segment D has preserved its identity, are purified.