Nucleic acid detection assays
AssigneeThird Wave Technologies, Inc.
InventorsJames R. Prudent, Jeff G. Hall, Victor I. Lyamichev, Mary Ann D. Brow, James E. Dahlberg
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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 forming 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.
Claims40
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 source of target nucleic acid, said 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 a first portion of said first oligonucleotide comprises at least one nucleotide analog and wherein said first portion is completely complementary to said first portion of said first target nucleic acid, and;
- iv) a second oligonucleotide comprising a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second region of said target nucleic acid;
- b) combining said cleavage agent, said target nucleic acid, said first oligonucleotide and said second oligonucleotide under reaction conditions such that at least said first 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 and cleaves said first oligonucleotide to generate a non-target cleavage product;
- and
- c) detecting the cleavage of said cleavage structure.
A method of detecting a target nucleic acid, comprising:
- a) providing:
- i) a cleavage agent;
- ii) a sample suspected of containing a target nucleic acid;
- iii) a first oligonucleotide comprising a 5′ portion complementary to a first region of said target nucleic acid;
- and
- iv) a second oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion;
- wherein at least one of said first oligonucleotide, said second oligonucleotide or said first or said second regions of said target nucleic acid comprises a nucleotide analog;
- b) combining said cleavage agent, said sample suspected of containing a target nucleic acid, said first oligonucleotide and said second oligonucleotide under reaction conditions such that at least said first 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 a non-target cleavage product;
- and
- c) detecting the cleavage of said cleavage structure.
A method for detecting a target sequence, comprising cleaving a cleavage structure with a cleavage agent to generate a labeled cleavage product, wherein said cleavage structure comprises:
- g) a first nucleic acid molecule comprising a first region and a second region, said first region upstream of said second region;
- h) a second nucleic acid molecule that is complementary to said first region, said second nucleic acid molecule having a 3′ end that is not extendable by a polymerase when said second nucleic acid molecule is hybridized to said first nucleic acid molecule;
- and
- i) a third nucleic acid molecule that is complementary to said second region, said third nucleic acid molecule comprising a label, wherein said labeled cleavage product comprises said label following cleavage of said cleavage structure.
The method of claim 36, wherein said cleavage agent comprises a 5′ nuclease.
Dependencies are inferred from the claim text.