Nucleic acid cleavage assays

Abstract

The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

1 . A method for detecting a target nucleic acid, comprising: 
 a) generating a cleavage structure that is cleavable by Mja FEN-1 endonuclease, wherein said cleavage structure comprises: 
 i. said target nucleic acid; and  
 ii. a displaced 5′ nucleic acid arm;  
   b) cleaving said cleavage structure with a thermostable 5′ nuclease that lacks synthetic activity to release said displaced 5′ nucleic acid arm; and    c) detecting the cleavage of said cleavage structure to detect said target nucleic acid.    
     
     
         2 . The method of  claim 1 , wherein a plurality of said cleavage structures are formed and cleaved.  
     
     
         3 . The method of  claim 2 , wherein a plurality of cleavage structures are formed on a single copy of said target nucleic acid.  
     
     
         4 . The method of  claim 2 , wherein said plurality of cleavage structures are formed and cleaved under isothermal conditions.  
     
     
         5 . The method of  claim 1 , wherein said displaced 5′ nucleic acid arm comprises are region that is complementary to said target nucleic acid.  
     
     
         6 . The method of  claim 5 , wherein said thermostable 5′ nuclease comprises a FEN-1 endonuclease.  
     
     
         7 . The method of  claim 5 , wherein said thermostable 5′ nuclease comprises a polymerase having 5′ nuclease activity.  
     
     
         8 . The method of  claim 7 , wherein said polymerase is derived from an organism of the genus  Thermus.    
     
     
         9 . The method of  claim 8 , wherein said organism comprises  Thermus aquaticus.    
     
     
         10 . The method of  claim 1 , wherein said target nucleic acid comprises a nucleic acid isolated from a biological sample.  
     
     
         11 . The method of  claim 10 , wherein said biological sample comprises an infectious disease organism.  
     
     
         12 . The method of  claim 1 , wherein said target nucleic acid comprises DNA.  
     
     
         13 . The method of  claim 1 , wherein said target nucleic acid comprises RNA.  
     
     
         14 . The method of  claim 1 , wherein said target nucleic acid comprises a synthetic nucleic acid molecule.  
     
     
         15 . The method of  claim 14 , wherein said synthetic nucleic acid molecule is generated in a polymerase chain reaction.  
     
     
         16 . The method of  claim 1 , wherein at least one nucleic acid molecule in said cleavage structure comprises a nucleotide analog.  
     
     
         17 . The method of  claim 16 , wherein said non-natural nucleotide comprises a degenerate nucleotide.  
     
     
         18 . The method of  claim 1 , wherein said cleavage structure comprises said target nucleic acid hybridized to a probe nucleic acid, said probe nucleic acid comprising said displaced 5′ nucleic acid arm.  
     
     
         19 . The method of  claim 18 , wherein said probe nucleic acid comprises a label.  
     
     
         20 . The method of  claim 19 , wherein said label comprises a fluorescent label.  
     
     
         21 . The method of  claim 18 , wherein said cleavage structure further comprises an upstream nucleic acid molecule hybridized to said target nucleic acid, said upstream nucleic acid molecule having a 3′ moiety that overlaps with a region of the target nucleic that is hybridized to said probe nucleic acid.  
     
     
         22 . The method of  claim 21 , wherein said 3′ moiety of said upstream nucleic acid molecule comprises a nucleotide.  
     
     
         23 . The method of  claim 22 , wherein said nucleotide is complementary to said target nucleic acid.  
     
     
         24 . The method of  claim 22 , wherein said 3′ moiety of said upstream nucleic acid molecule comprises a 3′ terminal nucleotide that is not complementary to said target nucleic acid.  
     
     
         25 . The method of  claim 22 , wherein said 3′ moiety of said upstream nucleic acid molecule consists of a 3′ terminal nucleotide that is not complementary to said target nucleic acid.  
     
     
         26 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence.  
     
     
         27 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection of mass.  
     
     
         28 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence energy transfer.  
     
     
         29 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.  
     
     
         30 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises directly detecting a nucleic acid fragment generated by cleavage of said cleavage structure.  
     
     
         31 . The method of  claim 30 , wherein said fragment comprises said displaced 5′ nucleic acid arm.  
     
     
         32 . The method of  claim 30 , wherein said fragment comprises two or more nucleotides.  
     
     
         33 . The method of  claim 1 , wherein said detecting the cleavage of said cleavage structure comprises indirectly detecting a nucleic acid fragment generated by cleavage of said cleavage structure.  
     
     
         34 . The method of  claim 33 , wherein said indirectly detecting comprises hybridizing said nucleic acid fragment to a template nucleic acid to form a detection complex.  
     
     
         35 . The method of  claim 34 , wherein said detection complex is configured to be bound by a protein.  
     
     
         36 . The method of  claim 35 , wherein said protein is a ligase.  
     
     
         37 . The method of  claim 35 , wherein said protein is a polymerase.  
     
     
         38 . The method of  claim 36 , wherein said polymerase is a DNA polymerase.  
     
     
         39 . The method of  claim 36 , wherein said polymerase is an RNA polymerase.  
     
     
         40 . The method of  claim 1 , wherein at least one nucleic acid molecule in said cleavage structure is attached to a solid support.  
     
     
         41 . A method for detecting a target nucleic acid, comprising: 
 a) generating a cleavage structure that is cleavable by Mja FEN-1 endonuclease, wherein said cleavage structure comprises: 
 iii. said target nucleic acid; and  
 iv. a displaced 5′ nucleic acid arm;  
   b) cleaving said cleavage structure with a thermostable FEN-1 nuclease to release said displaced 5′ nucleic acid arm; and    c) detecting the cleavage of said cleavage structure to detect said target nucleic acid.    
     
     
         42 . The method of  claim 41 , wherein a plurality of said cleavage structures are formed and cleaved.  
     
     
         43 . The method of  claim 42 , wherein a plurality of cleavage structures are formed on a single copy of said target nucleic acid.  
     
     
         44 . The method of  claim 42 , wherein said plurality of cleavage structures are formed and cleaved under isothermal conditions.  
     
     
         45 . The method of  claim 41 , wherein said target nucleic acid comprises a nucleic acid isolated from a biological sample.  
     
     
         46 . The method of  claim 45 , wherein said biological sample comprises an infectious disease organism.  
     
     
         47 . The method of  claim 41 , wherein said target nucleic acid comprises DNA.  
     
     
         48 . The method of  claim 41 , wherein said target nucleic acid comprises RNA.  
     
     
         49 . The method of  claim 41 , wherein said target nucleic acid comprises a synthetic nucleic acid molecule.  
     
     
         50 . The method of  claim 49 , wherein said synthetic nucleic acid molecule is generated in a polymerase chain reaction.  
     
     
         51 . The method of  claim 41 , wherein at least one nucleic acid molecule in said cleavage structure comprises a nucleotide analog.  
     
     
         52 . The method of  claim 51 , wherein said nucleotide analog comprises a degenerate nucleotide.  
     
     
         53 . The method of  claim 41 , wherein said cleavage structure comprises said target nucleic acid hybridized to a probe nucleic acid, said probe nucleic acid comprising said displaced 5′ nucleic acid arm.  
     
     
         54 . The method of  claim 53 , wherein said probe nucleic acid comprises a label.  
     
     
         55 . The method of  claim 54 , wherein said label comprises a fluorescent label.  
     
     
         56 . The method of  claim 53 , wherein said cleavage structure further comprises an upstream nucleic acid molecule hybridized to said target nucleic acid, said upstream nucleic acid molecule having a 3′ moiety that overlaps with a region of the target nucleic that is hybridized to said probe nucleic acid.  
     
     
         57 . The method of  claim 56 , wherein said 3′ moiety comprises a nucleotide.  
     
     
         58 . The method of  claim 56 , wherein said 3′ moiety of said upstream nucleic acid molecule comprises a nucleotide that is complementary to said target nucleic acid.  
     
     
         59 . The method of  claim 56 , wherein said 3′ moiety of said upstream nucleic acid molecule comprises a 3′ terminal nucleotide that is not complementary to said target nucleic acid.  
     
     
         60 . The method of  claim 56 , wherein said 3′ moiety of said upstream nucleic acid molecule consists of a 3′ terminal nucleotide that is not complementary to said target nucleic acid.  
     
     
         61 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence.  
     
     
         62 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises detection of mass.  
     
     
         63 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence energy transfer.  
     
     
         64 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.  
     
     
         65 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises directly detecting a nucleic acid fragment generated by cleavage of said cleavage structure.  
     
     
         66 . The method of  claim 65 , wherein said fragment comprises said displaced 5′ nucleic acid arm.  
     
     
         67 . The method of  claim 65 , wherein said fragment comprises two or more nucleotides.  
     
     
         68 . The method of  claim 41 , wherein said detecting the cleavage of said cleavage structure comprises indirectly detecting a nucleic acid fragment generated by cleavage of said cleavage structure.  
     
     
         69 . The method of  claim 68 , wherein said indirectly detecting comprises hybridizing said nucleic acid fragment to a template nucleic acid to form a detection complex.  
     
     
         70 . The method of  claim 69 , wherein said detection complex is configured to be bound by a protein.  
     
     
         71 . The method of  claim 70 , wherein said protein is a ligase.  
     
     
         72 . The method of  claim 70 , wherein said protein is a polymerase.  
     
     
         73 . The method of  claim 72 , wherein said polymerase is a DNA polymerase.  
     
     
         74 . The method of  claim 72 , wherein said polymerase is an RNA polymerase.  
     
     
         75 . The method of  claim 41 , wherein at least one nucleic acid molecule in said cleavage structure is attached to a solid support.