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 composition comprising a purified thermostable FEN-1 endonuclease and a polymerase.
27 . The composition of claim 26 , wherein said thermostable FEN-1 endonuclease comprises a FEN-1 endonuclease from an archaebacterial species.
28 . The composition of claim 27 , wherein said FEN-1 endonuclease comprises a FEN-1 endonuclease from Pyrococcus furiosus.
29 . The composition of claim 26 , wherein said FEN-1 endonuclease comprises the sequence SEQ ID NO: 115.
30 . The composition of claim 27 , wherein said FEN-1 endonuclease comprises a FEN-1 endonuclease from Methanococcus jannaschii.
31 . The composition of claim 26 , wherein said FEN-1 endonuclease comprises the sequence SEQ ID NO: 111.
32 . The composition of claim 26 , wherein said thermostable FEN-1 endonuclease comprises a non-natural thermostable FEN-1 endonuclease.
33 . The composition of claim 32 , wherein said non-natural thermostable FEN-1 endonuclease comprises a chimerical FEN-1 endonuclease.
34 . The composition of claim 26 , wherein said polymerase comprises a thermostable polymerase.
35 . The composition of claim 26 , wherein said polymerase comprises a template-independent polymerase.
36 . The composition of claim 26 , wherein said polymerase comprises a template-dependent polymerase.
37 . The composition of claim 26 , wherein said purified thermostable FEN-1 endonuclease and said polymerase are in a mixture.
38 . The composition of claim 26 , wherein said mixture comprises a reaction mixture.
39 . The composition of claim 26 , further comprising a nucleic acid molecule.
40 . The composition of claim 39 , wherein said nucleic acid molecule comprises a probe oligonucleotide.
41 . The composition of claim 39 , wherein said nucleic acid molecule is attached to a solid support.
42 . The composition of claim 26 , further comprising a buffer comprising magnesium.
43 . The composition of claim 26 , further comprising a labeled molecule.
44 . The composition of claim 43 , wherein said labeled molecule comprises a labeled nucleic acid molecule.
45 . The composition of claim 44 , wherein said labeled nucleic acid molecule comprises a fluorescent label.
46 . The composition of claim 26 , further comprising first and second nucleic acid molecules.
47 . The composition of claim 46 , 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.
48 . The composition of claim 47 , further comprising a stacker oligonucleotide.