Cleavage of nucleic acids
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.
1 - 25 . (canceled)
26 . A method for detecting a target nucleic acid comprising exposing a composition comprising a purified thermostable FEN-1 endonuclease and a polymerase to a sample suspected of containing a target nucleic under conditions such that said target nucleic acid, if present in said sample, is detected.
27 . The method of claim 26 , wherein said thermostable FEN-1 endonuclease comprises a FEN-1 endonuclease from an archaebacterial species.
28 . The method of claim 27 , wherein said archaeal FEN-1 endonuclease comprises a FEN-1 endonuclease from Pyrococcus furiosus.
29 . The method of claim 26 , wherein said FEN-1 endonuclease comprises the sequence SEQ ID NO:115.
30 . The method of claim 27 , wherein said FEN-1 endonuclease comprises a FEN-1 endonuclease from Methanococcus jannaschii.
31 . The method of claim 26 , wherein said FEN-1 endonuclease comprises the sequence SEQ ID NO:111.
32 . The method of claim 26 , wherein said thermostable FEN-1 endonuclease comprises a non-natural thermostable FEN-1 endonuclease.
33 . The method of claim 32 , wherein said non-natural thermostable FEN-1 endonuclease comprises a chimerical FEN-1 endonuclease.
34 . The method of claim 26 , wherein said polymerase comprises a thermostable polymerase.
35 . The method of claim 26 , wherein said polymerase comprises a template-independent polymerase.
36 . The method of claim 26 , wherein said polymerase comprises a template-dependent polymerase.
37 . The method of claim 26 , wherein said purified thermostable FEN-1 endonuclease and said polymerase are in a mixture.
38 . The method of claim 26 , wherein said mixture comprises a reaction mixture.
39 . The method of claim 26 , wherein said exposing comprises hybridizing a nucleic acid molecule to said target nucleic acid.
40 . The method of claim 39 , wherein said nucleic acid molecule comprises a probe oligonucleotide.
41 . The method of claim 39 , wherein said nucleic acid molecule is attached to a solid support.
42 . The method of claim 39 , wherein said nucleic acid molecule comprises a label.
43 . The method of claim 42 , wherein said label comprises a fluorescent label.
44 . The method of claim 26 , wherein said exposing comprises hybridizing first and second nucleic acid molecules to said target nucleic acid.
45 . The method of claim 44 , wherein said first and second nucleic acid molecules are capable of hybridizing to a target nucleic acid having a first region and a second region, said second region downstream of and contiguous to said first region, wherein at least a portion of said first nucleic acid molecule is completely complementary to said first region of said target nucleic acid and wherein said second nucleic acid molecule comprises a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second region of said target nucleic acid.
46 . The method of claim 44 , further comprising hybridizing a stacker oligonucleotide to said target nucleic acid.
47 . The method of claim 26 , wherein said detecting comprises detecting a non-target cleavage product.
48 . The method of claim 45 , wherein said 3′ portion of said second nucleic acid molecule consists of a single nucleotide not complementary to said target nucleic acid.
49 . The method of claim 45 , wherein said 3′ portion of said second nucleic acid molecule comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
50 . The method of claim 45 , wherein said 3′ portion of said second nucleic acid molecule comprises a 3′ terminal nucleotide complementary to said target nucleic acid.
51 . The method of claim 45 , wherein said 3′ portion of said second nucleic acid molecule comprises a single nucleotide.
52 . The method of claim 45 , wherein a 3′ terminus of said 3′ portion of said second nucleic acid molecule comprises an aromatic ring structure that is not a base.
53 . The method of claim 45 , wherein said first oligonucleotide further comprises a 5′ terminal portion that is not complementary to said target nucleic acid.
54 . The method of claim 26 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, and fluorescence polarization.
55 . The method of claim 26 , wherein said detecting the cleavage of said cleavage structure comprises detection based on the charge of non-target cleavage product.
56 . The method of claim 26 , wherein said target nucleic acid is DNA.
57 . The method of claim 26 , wherein said target nucleic acid is synthetic nucleic acid.
58 . The method of claim 57 , wherein said synthetic nucleic acid comprises amplified nucleic acid.
59 . The method of claim 58 , wherein said synthetic nucleic acid is generated by a polymerase chain reaction.
60 . The method of claim 26 , wherein said target nucleic acid is RNA.