Patent ID: 7682791

Claim:
A method for generating a nested set of double stranded DNA circles, the method comprising the steps of: providing one or more double stranded DNA circles each comprising an adaptor and a DNA fragment separate from the adaptor, the adaptor containing a recognition site for a restriction endonuclease that has a cleavage site located a predetermined distance within the DNA fragment and that produces cleavage products having overhanging strands; and combining in a reaction mixture the one or more double stranded DNA circles under conditions such that the following enzymatic activities are present: (i) a restriction endonuclease activity recognizing the restriction site in the adaptor, which cleaves the DNA fragment of the double stranded DNA circles to produce linear DNA molecules having ends with overhanging strands, (ii) a polishing enzyme activity that removes overhanging strands from linear DNA molecules to produce blunt-end linear DNA molecules, and (iii) a ligase activity that forms closed double stranded DNA circles from linear blunt-end DNA molecules, whereby a cyclical reaction takes place to generate a nested set of shortened double stranded DNA circles in the reaction mixture.