Polymerase driven nesa

A method for detecting a target polynucleotide, wherein the target polynucleotide comprises a target sequence flanked by a first hybridization sequence and a second hybridization sequence, the method comprising

• (a) exposing the target polynucleotide to an initiating oligonucleotide comprising (i) a 5′ end sequence complementary to the first hybridization sequence, (ii) a 3′ end sequence complementary to the second hybridization sequence, and (iii) a DNA synthesis primer recognition sequence, whereby a complex of the target polynucleotide and the initiating oligonucleotide is formed, wherein the complex comprises a first hybridization region formed between the 5′ end sequence and the first hybridization sequence and a second hybridization region formed between the 3′ end sequence and the second hybridization sequence;
• (b) extending the initiating oligonucleotide in the complex with an extended sequence, whereby the extended sequence is complementary to the target sequence;
• (c) ligating the initiating oligonucleotide sequence with the extended sequence to form a circular oligonucleotide, wherein the circular oligonucleotide comprises a nicking endonuclease (NE) recognition/cutting sequence;
• (d) exposing the circular oligonucleotide to a DNA polymerase and a DNA synthesis primer having a sequence complementary to the DNA synthesis primer recognition sequence, whereby a DNA is synthesized, wherein the synthesized DNA comprises a sequence corresponding to the target sequence, a sequence complementary to the DNA synthesis primer recognition sequence, and a NE recognition sequence complementary to the NE recognition/cutting sequence;
• (e) exposing the synthesized DNA to a probe having the NE recognition/cutting sequence, whereby a double stranded DNA comprising the probe and the synthesized DNA is formed, and wherein the double stranded DNA comprises a full NE recognition site formed by the NE recognition/cutting sequence and the NE recognition sequence;
• (f) exposing the double stranded DNA to a nicking endonuclease (NE) capable of recognizing the full NE recognition site and cleaving the NE recognition/cutting sequence in the full NE recognition site, whereby the probe in the double stranded DNA is cleaved and the synthesized DNA is dissociated from the probe after the cleavage, and whereby the dissociated synthesized DNA comprises a single copy of the target sequence;
• and
• (g) detecting the cleaved probe;
• wherein the presence of the cleaved probe indicates the presence of the target polynucleotide.