Method for engineering strand-specific, sequence-specific, DNA-nicking enzymes
US7081358B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Aug 16, 2002 |
| Grant date | Jul 25, 2006 |
| Priority date | — |
| Expiry date | Jan 6, 2023 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N9/22
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.