Patent ID: 7678541

Claim:
A method of detecting a target nucleic acid, comprising (a) providing: a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region, a template nucleic acid, which comprises in 3′ to 5′ order a first region and a second region, a first oligonucleotide that is at least partially complementary to said first region of said target nucleic acid, a second oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is complementary to said second region of said target nucleic acid and wherein said 5′ region is not complementary to said target nucleic acid but is at least partially complementary to said first region of said template nucleic acid, a third oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is at least partially complementary to said second region of said template nucleic acid and said 5′ region is not complementary to said template nucleic acid, a cleavage agent, and a polymerization agent; (b) mixing said target nucleic acid, said template nucleic acid, said first oligonucleotide, said second oligonucleotide, said third oligonucleotide, said cleavage agent and said polymerization agent under reaction conditions, wherein said reaction conditions permit: forming a first duplex between said target nucleic acid and each of said fast oligonucleotide and said second oligonucleotide, wherein the 5′ region of the second oligonucleotide forms a first flap of a first non-invasive cleavage structure, wherein the complementary portions of said first oligonucleotide and said second oligonucleotide are separated by at least a nick when the fast duplex is formed, cleaving the fast flap by said cleavage agent, thereby permitting release of the first flap, forming a second duplex with the released fast flap, said template nucleic acid, and said third oligonucleotide, wherein the 5′ region of the third oligonucleotide forms a second flap of a second non-invasive cleavage structure, wherein the complementary portions of said first flap and said third oligonucleotide are separated by at least a nick when said second duplex is formed, and cleaving the second flap by said cleavage agent; and (c) detecting the non-invasive cleavage of the second flap, wherein detection of said non-invasive cleavage of said second flap is indicative of the presence of said target nucleic acid.