Method for molecular cloning and polynucleotide synthesis...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/11 (2006.01) C12N 9/90 (2006.01) C12N 15/09 (2006.01) C12N 15/10 (2006.01) C12N 15/66 (2006.01) C12N 15/90 (2006.01) C12P 19/34 (2006.01)

Patent

CA 2208062

This invention provides a modified vaccinia topoisomerase enzyme containing an affinity tag which is capable of facilitating purification of protein-DNA complexes away from unbound DNA. This invention further provides a modified sequence specific topoisomerase enzyme. This invention provides a method of ligating duplex DNAs, a method of molecular cloning of DNA, a method of synthesizing polynucleotides, and a method of gene targeting. Lastly, this invention provides a recombinant DNA molecule composed of segments of DNA which have been joined ex vivo by the use of a sequence specific topoisomerase and which has the capacity to transform a suitable host cell comprising a DNA sequence encoding polypeptide activity.

L'invention concerne une enzyme modifiée de topoisomérase de vaccine contenant un marquage d'affinité capable de faciliter la purification de complexes de protéines et d'ADN à distance de l'ADN non fixée. L'invention concerne, de plus, une enzyme modifiée de topoisomérase spécifique de séquences. Elle concerne également un procédé de ligature d'ADN duplex, un procédé de clonage moléculaire d'ADN, un procédé de synthèse de polynucléotides et un procédé de ciblage de gènes. Enfin, elle concerne une molécule d'ADN recombiné composée de segments d'ADN réunis ex vivo au moyen d'une topoisomérase spécifique de séquences, et capable de transformer une cellule hôte appropriée comprenant une séquence d'ADN codant une activité de polypeptide.

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