Reactions on a solid surface
View Patent ↗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 solid support 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 characterizing a nucleic acid comprising:
a) providing:
i. a sample suspected of containing a target nucleic acid;
ii. oligonucleotides capable of hybridizing to said target nucleic acid to form an invasive cleavage structure, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid;
iii. a solid support, wherein one or more of said oligonucleotides is attached to said solid support; and
iv. an archaeal FEN-1 endonuclease active at 55° C. and above for detecting the presence of an invasive cleavage structure;
b) exposing said sample to said oligonucleotides and said FEN-1 endonuclease, wherein said exposing said sample to said oligonucleotides and said FEN-1 endonuclease comprises exposing said sample to said oligonucleotides and said FEN-1 endonuclease under conditions wherein an invasive cleavage structure is formed between a target nucleic acid and said oligonucleotides if said target nucleic acid is present in said sample; and
c) detecting said invasive cleavage structure.
2. The method of claim 1 , wherein one or more of said first oligonucleotides and/or one or more of said second oligonucleotides is attached to said solid support.
3. The method of claim 1 , wherein said first oligonucleotides comprise a fluorescent dye.
4. The method of claim 3 , wherein said first oligonucleotides further comprise a quencher molecule.
5. The method of claim 1 , further providing a spacer molecule, wherein said one or more oligonucleotides is attached to said solid support through said spacer molecule.
6. The method of claim 5 , wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.
7. The method of claim 1 , wherein said solid support comprises a support selected from the group consisting of a glass solid support, a plastic solid support, a silicon solid support, a latex solid support and a hydrogel solid support.
8. The method of claim 1 , wherein said solid support comprises a solid support selected from the group consisting of a bead, a multi-well plate, a column, and a microarray.
9. The method of claim 1 , wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.
10. A method for cleaving multiple oligonucleotides comprising:
a) providing:
i. a plurality of first oligonucleotides attached to a solid support;
ii. a second oligonucleotide attached to said solid support; and
iii. a cleavage agent comprising an archaeal FEN-1 endonuclease active at 55° C. and above;
wherein said first oligonucleotides comprise a portion complementary to a first region of a target nucleic acid; and wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid;
b) exposing said solid support to said cleavage agent under conditions such that:
i. a first cleavage structure is formed, said first cleavage structure comprising one of said first oligonucleotides and said second oligonucleotide;
ii. said cleavage agent cleaves said first oligonucleotide in said first cleavage structure to produce a first cleavage fragment;
iii. after cleavage of said first oligonucleotide, a second cleavage structure is formed, said second cleavage structure comprising a second of said first oligonucleotides and said second oligonucleotide;
iv. said cleavage agent cleaves said first oligonucleotide in said second cleavage structure to produce a second cleavage fragment; wherein said conditions comprise exposing a target nucleic acid to said solid support; and
c) detecting the presence of said first or said second cleavage fragments.
11. The method of claim 10 , wherein said second oligonucleotide is attached to said solid support through a spacer molecule.
12. The method of claim 11 , wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with three or more of said first oligonucleotides.
13. The method of claim 12 , wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with ten or more of said first oligonucleotides.
14. The method of claim 11 , wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.
15. The method of claim 10 , wherein said solid support comprises a support selected from the group consisting of a glass solid support, a plastic solid support, a silicon solid support, a latex solid support and a hydrogel solid support.
16. The method of claim 10 , wherein said solid support comprises a solid support selected from the group consisting of a bead, a multi-well plate, a column, and a microarray.
17. The method of claim 10 , wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.