Dna manipulation methods, applications for synthetic enzymes...

C - Chemistry – Metallurgy – 12 – N

Patent

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Details

C12N 15/10 (2006.01) C12N 15/52 (2006.01) C12N 15/66 (2006.01) C12N 15/90 (2006.01) C12P 17/06 (2006.01) C12P 17/08 (2006.01)

Patent

CA 2376559

The invention comprises a method of assembling several DNA units in sequence in a DNA construct and all derivatives of this method. In particular the production of synthetic enzymes is contemplated. Each DNA unit is provided with the same restriction enzyme recognition site at its 5' and 3' ends. The restriction recognition site at its 3' end being combined with a recognition site for a DNA modification enzyme. A DNA construct having the same or a compatible accessible restriction site, as provided in the DNA unit, is cleaved at the restriction site by the appropriate restriction enzyme. The desired DNA unit is then inserted into the DNA construct, this ligated product subsequently being brought into contact with a DNA modification enzyme such that the restriction site at the 3' end of the inserted DNA unit is abolished. The ligated product is then cleaved at the remaining unmodified restriction recognition site and a subsequent DNA unit is inserted. This process is repeated introducing each desired DNA unit to give a DNA construct containing all the desired units in sequence.

L'invention concerne un procédé d'assemblage de plusieurs unités d'ADN en séquence dans une construction d'ADN et tous les dérivés de ce procédé. L'invention concerne, en particulier, la production d'enzymes synthétiques. Chaque unité d'ADN présente le même site de reconnaissance d'enzymes de restriction à leurs extrémités 5' et 3'. Ce site de reconnaissance d'enzymes de restriction est associé à son extrémité 3', au site de reconnaissance pour une enzyme de modification d'ADN. Une construction d'ADN présente le même site de restriction accessible ou un site de restriction compatible, tel que celui de l'unité d'ADN, clivé au site de restriction par l'enzyme de restriction appropriée. L'unité d'ADN désirée est ensuite insérée dans la construction d'ADN, ce produit ligaturé étant ensuite mis en contact avec une enzyme de modification d'ADN telle que le site de restriction à l'extrémité 3' de l'unité d'ADN insérée est abolie. Le produit ligaturé est ensuite clivé au site de reconnaissance de restriction non modifié restant et une autre unité d'ADN est insérée. Ce procédé est répété en introduisant chaque unité d'ADN désirée pour donner une construction d'ADN contenant toutes les unités désirées en séquence.

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