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 purified nucleic acid molecule.
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 26 , further comprising a polymerase.
34 . The composition of claim 33 , wherein said polymerase comprises a thermostable polymerase.
35 . The composition of claim 33 , wherein said polymerase comprises a template-independent polymerase.
36 . The composition of claim 33 , wherein said polymerase comprises a template-dependent polymerase.
37 . The composition of claim 26 , wherein said purified thermostable FEN-1 endonuclease and said purified nucleic acid molecule are in a mixture.
38 . The composition of claim 26 , wherein said nucleic acid molecule comprises a probe oligonucleotide.
39 . The composition of claim 26 , further comprising a buffer comprising magnesium.
40 . The composition of claim 26 , further comprising a labeled molecule.
41 . The composition of claim 26 , wherein said nucleic acid molecule comprises a label.
42 . The composition of claim 41 , wherein said label comprises a fluorescent label.
43 . The composition of claim 26 , further a second nucleic acid molecule.
44 . The composition of claim 43 , wherein said purified nucleic acid and said second nucleic acid molecule 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 purified 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.
45 . The composition of claim 44 , further comprising a stacker oligonucleotide.