End selection in directed evolution

C - Chemistry – Metallurgy – 12 – N

Patent

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C12N 15/10 (2006.01) C12N 9/14 (2006.01) C12N 15/55 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2361927

This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-sto- chastic methods of directed evolution (DirectEvolution.TM.). A particular advantage of end-selection-based methods is the ability to recover full-length polynucleotides from a library of progeny molecules by mutagenesis methods. These methods in- clude non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis.TM.) and non-stochastic polynu- cleotide reassembly (GeneReassembly.TM.). This invention provides methods of obtaining novel enzymes that have optmized physical and/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit ink efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased 4. The applicant believes it is entitled to claim status as a small entity status as defined under Section 2 of the Patent Rules. 5. The applicant accordingly hereby requests commencement of national phase procedures consequent to the designation ofCanada in such international application and prays that a patent may be granted to him for the said invention. SIGNED at Ottawa, Ontario, Canada this 28th day of August, 2001.

Cette invention concerne des techniques permettant d'obtenir des polynucléotides et des polynucléotides codés en utilisant des techniques non stochastiques d'évolution dirigées (DirectEvolution™). La possibilité de récupérer des polynucléotides de pleine longueur issus d'une bibliothèque de molécules d'une lignée générées par mutagenèse présente un avantage particulier dans les techniques fondées sur une sélection finale. Ces techniques comprennent des mutagenèses à saturation de site non stochastiques de polynucléotides (Gene Site Saturation Mutagenesis™) et un réassemblage de polynucléotides non stochastique (GeneReassembly™). Cette invention concerne des techniques permettant d'obtenir des enzymes aux propriétés physiques et/ou biologiques optimisées. Par l'utilisation de ces techniques de l'invention, on peut faire évoluer des vaccins génétiques, des enzymes, de petites molécules et d'autres molécules qui conviennent vers les propriétés souhaitées. On peut, par exemple, obtenir des vecteurs de vaccin qui font preuve d'une meilleure efficacité lorsqu'on les utilise comme vaccins génétiques. Les vecteurs ainsi obtenus peuvent avoir, par exemple, une expression antigénique renforcée, une absorption dans la cellule améliorée, une meilleure stabilité dans la cellule, une capacité à concevoir une réponse immune et d'autres propriétés de ce genre. Cette invention concerne en outre des techniques permettant d'obtenir diverses molécules biologiques actives, dans le domaine des antibiotiques, des pharmacothérapeutiques, et des caractères transgéniques.

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