Invasion assays
View Patent ↗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. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.
1. A method of detecting a target polynucleotide which comprises the steps of:
a) contacting a target polynucleotide having a first portion and a second portion immediately contiguous to one another with:
i) an invader oligonucleotide, at least a part of which is capable of specifically hybridizing to the first portion of the target polynucleotide;
ii) a first probe oligonucleotide comprising a first region that is capable of specifically hybridizing to the second portion of the target polynucleotide and an unpaired region located adjacent to the first region; and
iii) a reagent that is capable of cleaving to release the unpaired region of the first probe oligonucleotide to produce a cleaved unpaired region, wherein said reagent comprises a 5′ nuclease;
producing said cleaved unpaired region by cleaving said first probe oligonucleotide, forming a cleavage structure comprising said cleaved unpaired region and a second probe oligonucleotide by hybridizing said cleaved unpaired region to said second probe oligonucleotide, or forming a cleavage structure comprising said cleaved unpaired region, said second probe oligonucleotide, and a target nucleic acid by hybridizing said cleaved unpaired region and said second probe oligonucleotide to said target nucleic acid, and generating a cleaved second probe by cleaving said second cleavage structure using the reagent;
b) detecting the accumulation of the cleaved second probe oligonucleotide; and
c) determining whether the cleaved second probe oligonucleotide accumulates exponentially over time, wherein said exponential accumulation of the cleaved second probe oligonucleotide over time is indicative of the presence of said target polynucleotide.
2. The method of claim 1 wherein said detecting the accumulation of the cleaved second probe oligonucleotide comprises detection of fluorescence or phosphorescence.
3. The method of claim 1 , wherein said 5′ nuclease is a thermostable 5′ nuclease.
4. The method of claim 3 , wherein said thermostable 5′ nuclease is a FEN-1 nuclease.
5. The method of claim 4 , wherein said FEN-1 nuclease is a FEN-1 nuclease from an archaebacterial species.
6. The method of claim 5 , wherein said FEN-1 nuclease is selected from the group consisting of Methanococcus jannaschii FEN-1 and Pyrococcus furiosus FEN-1.
7. A method for detecting the presence of a first target nucleic acid in a sample, comprising:
a) incubating a sample containing a first target nucleic acid with a first nucleic acid molecule and a second nucleic acid molecule and forming a first cleavage structure, said first cleavage structure comprising:
i) said first target nucleic acid comprising a first region and a second region, said second region upstream of and contiguous to said first region;
ii) said first nucleic acid molecule comprising a first portion that is completely complementary the second region of the first target nucleic acid;
iii) said second nucleic acid molecule comprising a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said first region of said first target nucleic acid;
wherein said 5′ portion of said second nucleic acid molecule is annealed to said first region of said first target nucleic acid and wherein at least a portion of said first nucleic acid molecule is annealed to said second region of said first target nucleic acid, and wherein a 5′ portion of said first nucleic acid molecule is not annealed to said first target nucleic acid,
b) cleaving said first cleavage structure with a cleavage agent comprising a 5′ nuclease, generating a non-target cleavage product, and forming a second cleavage structure comprising:
i) said non-target cleavage product; and
ii) a probe oligonucleotide;
by hybridizing said non-target cleavage product to said probe oligonucleotide, or forming a second cleavage structure comprising:
i) said non-target cleavage product;
ii) said probe oligonucleotide; and
iii) a second target nucleic acid;
by hybridizing both said non-target cleavage product and said probe oligonucleotide to said second target nucleic acid, and
c) cleaving said second cleavage structure with said cleavage agent, generating a cleaved probe and detecting said cleaved probe at a plurality of time points, wherein said cleaved probe accumulates at an exponential rate over time, and wherein the accumulation of said cleaved probe at an exponential rate over time indicates the presence of said first target nucleic acid in said sample.
8. The method of claim 7 , wherein said detecting said cleaved probe comprises detection of fluorescence.
9. The method of claim 7 , wherein said detecting said cleaved probe comprises detection of mass.
10. The method of claim 7 , wherein said detecting said cleaved probe comprises detection of fluorescence energy transfer.
11. The method of claim 7 , wherein said detecting said cleaved probe comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
12. The method of claim 7 , wherein said 5′ nuclease is a thermostable 5′ nuclease.
13. The method of claim 12 , wherein said thermostable 5′ nuclease is a 5′ nuclease of a DNA polymerase.
14. The method of claim 13 , wherein said DNA polymerase is Taq DNA polymerase.
15. The method of claim 7 , wherein said 3′ portion of said second nucleic acid molecule consists of a single nucleotide.
16. The method of claim 15 , wherein said single nucleotide is complementary to said first target nucleic acid.
17. The method of claim 7 further comprising providing a plurality of said first nucleic acid molecule such that said first nucleic acid molecule is in concentration excess compared to said first target nucleic acid.
18. The method of claim 7 further comprising providing a plurality of said second nucleic acid molecule such that said second nucleic acid molecule is in concentration excess compared to said first target nucleic acid.
19. The method of claim 7 , wherein said first target nucleic acid and said second nucleic acid molecule form a duplex, and further comprising providing a plurality of said first nucleic acid molecule such that said first nucleic acid molecule is in concentration excess compared to said duplex.
20. The method of claim 19 , wherein said non-target cleavage product is generated from said first nucleic acid molecule and is in concentration excess compared to said duplex.
21. The method of claim 12 , wherein said thermostable 5′ nuclease is a FEN-1 nuclease.
22. The method of claim 21 , wherein said FEN-1 nuclease is a FEN-1 nuclease from an archaebacterial species.
23. The method of claim 22 , wherein said FEN-1 nuclease is selected from the group consisting of Methanococcus jannaschii FEN-1 and Pyrococcus furiosus FEN-1.