Systems for in vivo site-directed mutagenesis using...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/10 (2006.01) C07H 21/00 (2006.01) C12N 15/09 (2006.01) C12N 15/63 (2006.01) C12N 15/90 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2455013

This disclosure provides several methods to generate nucleic acid mutations in vivo, for instance in such a way that no heterologous sequence is retained after the mutagenesis is complete. The methods employ integrative recombinant oligonucleotides (IROs). Specific examples of the described mutagenesis methods enable site-specific point mutations, deletions, and insertions. Also provided are methods that enable multiple rounds of mutation and random mutagenesis in a localized region. The described methods are applicable to any organism that has a homologous recombination system.

Cette invention concerne plusieurs procédés qui permettent de réaliser des mutations d'acide nucléique in vivo de sorte, par exemple, qu'aucune séquence hétérologue ne soit retenue une fois la mutagenèse terminée. Les procédés de l'invention mettent en oeuvre des oligonucléotides recombinants d'intégration (IRO). Des exemples spécifiques des procédés de mutagenèse décrits permettent de réaliser des mutations, des délétions ou des insertions spécifiques d'un site. L'invention concerne en outre des procédés qui permettent de réaliser plusieurs cycles de mutation et une mutagenèse aléatoire dans une région localisée. Ces procédés sont applicables à tout organisme possédant un système de recombinaison homologue.

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