Patent ID: 8288095

Claim:
A method of polynucleotide sequencing, comprising: (a) providing a mixture of circular polynucleotides, wherein each of said circular polynucleotides comprises: (i) a first Type IIs restriction endonuclease recognition site; (ii) a target polynucleotide fragment; and (iii) a second Type IIs restriction endonuclease recognition site, wherein the first Type IIs restriction endonuclease recognition site is flanking a 5â€² end of the target polynucleotide fragment, the second Type IIs restriction endonuclease recognition site is flanking a 3â€² end of the target polynucleotide fragment, and the cleavage sites of the first and second Type IIs restriction endonucleases are within the interior of the target polynucleotide fragment, wherein the first and second Type IIs restriction endonuclease recognition sites are arranged such that a first Type IIs restriction endonuclease recognizing said first Type II restriction endonuclease recognition site and a second Type IIs restriction endonuclease recognizing said second Type II restriction endonuclease recognition site cleave the target polynucleotide fragment, and wherein said circular polynucleotides comprise different target polynucleotide fragments; (b) cleaving the different target polynucleotide fragments contained in said mixture of the circular polynucleotides with said first Type IIs restriction endonuclease and said second Type IIs restriction endonuclease, thereby producing restriction fragments comprising segments from the ends of the different target polynucleotide fragments; and (c) determining sequences of the segments from the ends of the different target polynucleotide fragments after the cleaving step.