Patent ID: 8137936

Claim:
A method of forming closed double stranded DNA circles from a plurality of target polynucleotides in a sample, the method comprising the steps of: providing one or more selection oligonucleotides for each of the plurality of target polynucleotide so that each of such one or more selection oligonucleotides has a phosphorylated 5′ end and is capable of specifically annealing at the same time to two non-contiguous sites on its respective target polynucleotide whenever present in the sample to form a circular complex therewith comprising a free 5′ strand and a free 3′ strand; and combining in a reaction mixture the sample, nucleoside triphosphates, and the selection oligonucleotides under conditions such that the following enzymatic activities are present: (i) a 5′ flap endonuclease activity, (ii) a DNA polymcrasc lacking strand displacement activity, (iii) a 3′ single stranded exonuclease activity, and (iv) a ligase activity, wherein for each circular complex any free 3′strand is digested to form an extendable duplex that is extended by the DNA polymerase activity to the free 5′ strand of the circular complex, any free 5′ strand of the circular complex adjacent to an extended extendable duplex is cleaved to form a first nick, the annealed selection oligonucleotide is extended from a 3′ end along the circular complex to a 5′end of the selection oligonucleotide to form a second nick, and the first and second nicks are ligated to form a closed double stranded DNA circle.