Method for making insertional mutations

Details Method for making insertional mutations Method for making insertional mutations

C - Chemistry, Metallurgy

12

N

C12N 15/90 (2006.01) C12N 9/22 (2006.01) C12N 15/10 (2006.01)

Patent

CA 2343000

A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises: (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed ~.nu.$g().omega.~.RHO.o under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

L'invention concerne une méthode permettant d'effectuer des mutations d'insertion sur des emplacements aléatoires ou quasi-aléatoires de l'acide nucléique chromosomique ou extra-chromosomique d'une cellule cible. Cette méthode consiste à combiner, dans la cellule cible, l'acide nucléique cellulaire avec un complexe synaptique qui comprend: (a) une protéine transposase Tn5 et (b) un polynucléotide comprenant une paire de séquences nucléotidiques adaptées pour interagir de manière fonctionnelle avec la transposase Tn5 et une séquence nucléotidique transposable intercalée, dans des conditions qui entraînent des transpositions dans l'ADN cellulaire. Dans cette méthode, le complexe synaptique est formé in vitro dans des conditions qui empêchent ou qui sont défavorables à une transposition productive des complexes synaptiques.

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