Invasive cleavage of nucleic acids
First Claim
26. A method for detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
- a) providing;
i) a cleavage agent;
ii) a synthetic target nucleic acid, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;
iii) a first oligonucleotide, wherein at least a portion of said first oligonucteotide is completely complementary to said first portion of said first target ntucleic acid;
iv) a second oligonucleotide comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second portion of said target nucleic acid;
b) mixing said cleavage agent, said synthetic target nucleic acid, said first oligonucleotide ind said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said portion of said first oligonucleotide is annealed to said first region of said target nucleic acid and wherein at least said 5′
portion of said second oligonucleotide is annealed to said second region of said target nucleic acid so as to create a cleavage structure, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage product; and
c) detecting the cleavage of said cleavage structure.
9 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for formning a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.
-
Citations
60 Claims
-
26. A method for detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
-
a) providing;
i) a cleavage agent;
ii) a synthetic target nucleic acid, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;
iii) a first oligonucleotide, wherein at least a portion of said first oligonucteotide is completely complementary to said first portion of said first target ntucleic acid;
iv) a second oligonucleotide comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second portion of said target nucleic acid;
b) mixing said cleavage agent, said synthetic target nucleic acid, said first oligonucleotide ind said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said portion of said first oligonucleotide is annealed to said first region of said target nucleic acid and wherein at least said 5′
portion of said second oligonucleotide is annealed to said second region of said target nucleic acid so as to create a cleavage structure, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage product; and
c) detecting the cleavage of said cleavage structure. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
-
-
43. A kit for detecting the presence of a synthetic target nucleic acid molecule, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region, the kit comprising:
-
a) a first oligonucleotide, wherein at least a portion of said first oligonucleotide is completely complementary to said first portion of said first target nucleic acid; and
b) a second oligonucleotide comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second portion of said target nucleic acid. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
-
-
60. A kit for detecting the presence of a synthetic target nucleic acid molecule comprising a FEN-1 endonuclease and amplification primers.
Specification