Methods and compositions for detecting target sequences
View Patent ↗The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention 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. For example, in some embodiments, a 5′ nuclease activity from any 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.
1. A method for detecting a target sequence, comprising:
a) providing a sample containing nucleic acid suspected of containing a target sequence;
b) generating an amplification product from said target nucleic acid, wherein said generating comprises formation of a circular nucleic acid by
i) annealing a probe oligonucleotide that forms a first invasive cleavage structure in the presence of said target sequence,
ii) cleaving said first invasive cleavage structure to form an annealed gapped structure,
iii) ligating said annealed gapped structure to form a circular nucleic acid;
c) treating said circular nucleic acid to amplification reaction conditions to produce an amplification product comprising no more than 8 contiguous bases that identically match said target sequence or its complement, and
d) exposing said amplification product to oligonucleotides capable of forming a second invasive cleavage structure with said amplification product and an agent for detecting the presence of said second invasive cleavage structure.
2. The method of claim 1 , wherein said agent comprises a cleavage agent.
3. The method of claim 1 , wherein said circular nucleic acid is amplified using rolling circle amplification.
4. The method of claim 1 , further comprising the step of e) detecting said cleavage of said second invasive cleavage structure.
5. The method of claim 1 , wherein said amplification product comprises a first region and a second region, said second region downstream of and contiguous to said first region, and wherein said oligonucleotides comprise first and second oligonucleotides, said wherein at least a portion of said first oligonucleotide is completely complementary to said first portion of said amplification product and wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second portion of said target nucleic acid.
6. The method of claim 5 , wherein said 3′ portion of said second oligonucleotide comprises a 3′ terminal nucleotide not complementary to amplification product.
7. The method of claim 6 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide not complementary to said amplification product.
8. The method of claim 4 , wherein said detecting the cleavage of said second invasive cleavage structure comprises detection selected from the group consisting of detection of fluorescence, mass, fluorescence energy transfer, radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
9. The method of claim 2 , wherein said cleavage agent comprises a structure-specific nuclease.
10. The method of claim 9 , wherein said structure-specific nuclease comprises a thermostable structure-specific nuclease.
11. The method of claim 1 , wherein said nucleic acid suspected of containing a target sequence is selected from the group consisting of DNA and RNA.
12. The method of claim 1 , wherein said nucleic acid suspected of containing a target sequence comprises viral nucleic acid.
13. The method of claim 12 , wherein said target nucleic acid comprises hepatitis C virus nucleic acid.