Patent ID: 6893819

Claim:
A method of producing detectable released flaps comprising (a) providing: one or more template nucleic acids, each of which comprises in 3′ to 5′ order a first region, an extension region, and a second region, an upstream primer that is at least partially complementary to said first region of said template nucleic acid, and one or more downstream oligonucleotides corresponding to said one or more template nucleic acids, each downstream oligonucleotide comprising a 5′ region and a 3′ region, wherein each said 3′ region is at least partially complementary to said second region of said corresponding template nucleic acid and wherein each said 5′ region is at least partially complementary to a region that may be upstream of, downstream of, or comprise said first region of said corresponding template nucleic acid; (b) mixing at least said template nucleic acid and said upstream primer and downstream oligonucleotide under conditions which permit formation of a duplex between said template nucleic acid and each of said upstream primer and said 3′ region of said downstream oligonucleotide, wherein said 5′ region of said downstream oligonucleotide is a flap; (c) subjecting said duplex to a nucleic acid polymerization activity under conditions which permit extension of said upstream primer by polymerization of a nucleic acid strand complementary to a length of said extension region sufficient to form a first cleavage structure; (d) providing a cleavage means under conditions such that cleavage of said first cleavage structure occurs at a site located within said downstream oligonucleotide in a manner dependent upon the formation of said first cleavage structure, thereby permitting cleavage and release of said flap of said downstream oligonucleotide; (e) permitting said released flap of said downstream oligonucleotide to associate with said template nucleic acid and; an uncleaved downstream oligonucleotide to form a second duplex comprising said template nucleic acid and each of said released flap of said downstream oligonucleotide and an uncleaved downstream oligonucleotide, wherein said 5′ region of said uncleaved downstream oligonucleotide is a second flap; (f) forming a second cleavage structure by subjecting said second duplex to a nucleic acid polymerization activity under conditions which permit extension of said released flap by polymerization of a nucleic acid strand complementary to a length of said extension region sufficient to form a second cleavage structure; (g) cleaving said second cleavage structure by providing a cleavage means under conditions such that cleavage of said second cleavage structure occurs at a site located within said uncleaved downstream oligonucleotide in a manner dependent upon the formation of said second cleavage structure, thereby permitting cleavage and release of said second flap; and (h) detecting released first and/or second flaps.