Methods and compositions for detecting target sequences
The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention 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. For example, in some embodiments, a 5′ nuclease activity from any 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. A method for detecting a target sequence, comprising:
a) providing:
i) a sample containing synthetic DNA amplified from genomic DNA, said genomic DNA suspected of containing said target sequence, wherein said synthetic DNA comprises a first region and a second region, said second region downstream of and contiguous to said first region;
ii) oligonucleotides capable of forming an invasive cleavage structure with said synthetic DNA when said genomic DNA contains said target sequence, wherein said oligonucleotides comprise first and second oligonucleotides, wherein at least a portion of said first oligonucleotide is completely complementary to said first region of said synthetic DNA and wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second region of said synthetic DNA; and
iii) an agent for cleaving an invasive cleavage structure, wherein said agent is a thermostable FEN nuclease; and
b) exposing said sample to said oligonucleotides and said agent under conditions wherein said oligonucleotides form an invasive cleavage structure with said synthetic DNA when said genomic DNA contains said target sequence, wherein said invasive cleavage structure is cleaved by said agent;
c) detecting cleavage of an invasive cleavage structure by said agent, thereby detecting the presence of said target sequence.
2. The method of claim 1 , wherein said genomic DNA is nicked double stranded genomic DNA, and wherein said synthetic DNA is amplified by extension of 3′ ends in said nicked double-stranded genomic DNA suspected of containing said target sequence.
3. The method of claim 1 , wherein said synthetic DNA amplified from genomic DNA is amplified by extension of multiple primers on said genomic DNA suspected of containing said target sequence.
4. The method of claim 3 , wherein said primers are random primers.
5. The method of claim 3 , wherein said primers are exonuclease resistant.
6. The method of claim 1 , wherein said 3′ portion of said second oligonucleotide comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
7. The method of claim 1 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide not complementary to said target nucleic acid.
8. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of fluorescence.
9. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of mass.
10. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of fluorescence energy transfer.
11. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
12. The method of claim 1 , wherein said synthetic DNA is attached to a solid support.
13. The method of claim 1 wherein said genomic DNA suspected of containing said target sequence is attached to a solid support.
14. The method of claim 1 , wherein said first oligonucleotide is attached to a solid support.
15. The method of claim 1 , wherein said second oligonucleotide is attached to a solid support.
16. The method of claim 1 , wherein a portion of the amino acid sequence of said thermostable FEN nuclease is homologous to a portion of the amino acid sequence of a thermostable FEN nuclease derived from a thermophilic organism.
17. The method of claim 16 , wherein said thermophilic organism is from an Archaebacterial species.
18. The method of claim 17 , wherein said Archaebacterial species is selected from the group consisting of Methanococcus jannaschii, Pyrococcus furiosus, Pyrococcus woesei, Methanobacterium thermoautotrophicum , and Archaeoglobus fulgidus.