Patent ID: 7309573

Claim:
A method of detecting a target nucleic acid, comprising (a) providing: said target nucleic acid, which comprises in 3′ to 5′ order a first region, an extension region, and a second region, a template nucleic acid, which comprises in 3′ to 5′ order a first region, an extension 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 at least partially 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 means, and a polymerization activity; (b) mixing said target nucleic acid, said template nucleic acid, said first oligonucleotide, said second oligonucleotide, said third oligonucleotide, said cleavage means and said polymerization activity under a series of reaction conditions, wherein said series of reaction conditions permit: formation of a duplex between said target nucleic acid and each of said first oligonucleotide and said 3′ region of said second oligonucleotide, wherein said 5′ region of said second oligonucleotide is a flap, extension of said first oligonucleotide by polymerization of a nucleic acid strand complementary to a length of said extension region of said target nucleic acid sufficient to form a first cleavage structure, cleavage of said first cleavage structure within said second oligonucleotide by said cleavage means, thereby permitting release of said flap of said second oligonucleotide, formation of a second duplex with said released flap of said second oligonucleotide, said template nucleic acid, and said third oligonucleotide, wherein said 5′ region of said third oligonucleotide is a second flap, extension of said released flap by polymerization of a nucleic acid strand complementary to a length of said extension region of said template nucleic acid sufficient to form a second cleavage structure, and cleavage of said second cleavage structure within said third oligonucleotide by said cleavage means, thereby permitting cleavage and release of said third oligonucleotide; and (c) detection of a signal generated by said release of said third oligonucleotide from said template nucleic acid.