Centre National De La Recherche Scientifique

Florence Boe

Jean-Michel Masson

This procedure comprises the following steps: (1) cleavage of a circular cloning vector comprising two restriction sites a and b giving cohesive ends which are compatible with one another, and two restriction sites c and d which are close to the restriction sites a and b, by restriction enzyme(s), (2) introduction by ligation of the nucleic acid sequence to be polymerized into the linearized vector obtained in (1), between the two restriction sites a and b (1x vector), (3) cleavage of the recircularized vector obtained in (2), by restriction enzyme(s), (4) introduction by ligation of a gene coding for a suppressor tRNA into the linearized vector obtained in (3), between the two restriction sites c and d (1x+s vector), (5) cleavage of the vector obtained in (4) by restriction enzyme(s), (6) introduction by ligation of the fragment a-d obtained in (5) (1x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector at the said sites b and d, (7) introduction by ligation of the fragment obtained in (6) (2x+s fragment) between the sites b and d of a 1x vector as obtained in (2), after cleavage of this 1x vector, the cleavage being as defined in (6), (8) repetition of step (7), by insertion of the nx+s fragment into a 1x vector, until a sequence containing n+1 fragments is obtained, (9) transformation of a bacterial strain with the vector obtained in (8), and selection of mut+ strains, and (10) extraction of the (n+1)x polymer from the said selected strains.

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Toulouse, France

Florence Boe