The invention provides a method of generating nested sets of double stranded DNA (dsDNA) circles that may be used as size ladders in nucleic acid separations and as templates in DNA sequencing operations. In one aspect, the invention provides methods for generating nested sets of double stranded DNA circles in a self-sustaining enzymatic reaction comprising the activities of at least one endonuclease, at least one single stranded exonuclease, and at least one ligase. In another embodiment, such nested sets are generated from linear dsDNA fragments having ligatable terminators that are self-ligated to form corresponding dsDNA circles.