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 cleaving multiple oligonucleotides comprising:
a) providing:
i. a plurality of first oligonucleotides attached to a single solid support;
ii. a second oligonucleotide attached to the same solid support as said plurality of first oligonucleotides;
iii. a target nucleic acid, wherein said target nucleic acid comprises a first region and a second region, said second region downstream of and contiguous to said first region, wherein at least a portion of each of said first oligonucleotides is completely complementary to said first region of said target nucleic acid, 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 target nucleic acid; and
iv. a cleavage agent comprising a FEN-1 endonuclease from an archaebacterial species;
and
b) exposing said solid support to said cleavage agent under conditions such that:
i. a first invasive cleavage structure is formed, said first invasive cleavage structure comprising a first one of said plurality of first oligonucleotides, said second oligonucleotide, and said target nucleic acid;
ii. said cleavage agent cleaves said first one of said plurality of first oligonucleotides in said first invasive cleavage structure to produce a first cleavage fragment;
iii. after cleavage of said first one of said plurality of first oligonucleotides, a second invasive cleavage structure is formed, said second invasive cleavage structure comprising a second one of said plurality of first oligonucleotides and the same second oligonucleotide as was used in said first invasive cleavage structure;
iv. said cleavage agent cleaves said first oligonucleotide in said second cleavage structure to produce a second cleavage fragment.
2. The method of claim 1 , further comprising the step of c) detecting the presence of said first or said second cleavage fragments.
3. The method of claim 1 , wherein said second oligonucleotide is attached to said solid support through a spacer molecule.
4. The method of claim 3 , wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with three or more of said plurality of first oligonucleotides.
5. The method of claim 4 , wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with ten or more of said plurality of first oligonucleotides.
6. The method of claim 3 , 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 glass solid support.
8. The method of claim 1 , wherein said solid support comprises a latex solid support.
9. The method of claim 1 , wherein said solid support comprises a hydrogel solid support.
10. The method of claim 1 , wherein said solid support comprises a bead.
11. The method of claim 1 , wherein said solid support comprises a multi-well plate.
12. The method of claim 1 , wherein said solid support comprises a column.
13. The method of claim 1 , wherein said solid support comprises a microarray.
14. The method of claim 1 , wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.