Patent ID: 7901890
Filing Date: 2011-03-08
Classification: C12Q

Abstract:
1. A method for selectively activating a recognition site for a Type IIs restriction endonuclease in a nucleic acid sequence, the method comprising: (a) providing a nucleic acid sequence comprising first and second recognition sites for a Type IIs restriction endonuclease; (b) amplifying the nucleic acid sequence using a uracil-containing primer that has a sequence that is complementary to the first recognition site, thereby producing an amplified nucleic acid sequence comprising a first recognition site for a Type IIs restriction endonuclease comprising, one or more uracils at or near the first recognition site, and a second recognition site for a Type IIs restriction endonuclease; (c) degrading said one or more uracils at or near the first recognition site, thereby producing a single-stranded region in the first recognition site and protecting the first recognition site from methylation by a methylase that methylates unprotected recognition sites for the Type IIs restriction endonuclease; (d) methylating the second recognition site with the methylase, inhibiting digestion of the nucleic acid sequence by the Type IIs restriction endonuclease resulting from recognition of the second recognition site; and (e) making the single-stranded region double-stranded such that the Type IIs restriction endonuclease can recognize the first recognition site and digest the nucleic acid sequence.