Signal confinement sequencing (SCS) and nucleotide analogues for signal confinement sequencing
Novel fluorescent nucleotide analogs are provided herein. Also provided herein are methods of using the nucleotide analogs in sequencing-by-synthesis and signal confinement methods.
1. A method for sequencing a target nucleic acid, comprising:
a) providing a template nucleic acid immobilized on a surface, a primer, a polymerase, and a nucleotide analogue of the following structure:
wherein
n is 0, 1, 2, 3, 4, 5, 6, or 7;
L is absent or a linking group;
R 1 is a nucleoside base;
R 2 is hydrogen or a blocking group;
B is a binding molecule; and
X is a detection label, wherein said detection label is quenched;
b) extending the primer by incorporating the nucleotide analogue;
c) providing a phosphatase to cleave between the binding molecule and the linking group or between the binding molecule and the terminal phosphate, thereby generating a fragment of (I) comprising the label and the binding molecule, wherein the label is unquenched;
d) binding the fragment to a capture element immobilized on a surface; and
e) detecting a fluorescence emission from the label of the fragment captured on the surface.
2. The method of claim 1 , wherein the binding molecule comprises an oligonucleotide.
3. The method of claim 1 , wherein the capture element comprises a thio or thiol containing molecule.
4. The method of claim 1 , wherein the capture element comprises streptavidin, an antibody, a protein, or a dendrimer.
5. The method of claim 1 , wherein the capture element comprises an oligonucleotide immobilized complementary to the template nucleic acid.
6. The method of claim 1 , wherein the template nucleic acid is an immobilized DNA concatemer comprising multiple copies of an adaptor sequence.
7. The method of claim 1 , wherein L is a linking group and the linking group further comprises a quencher.
8. The method of claim 7 , wherein the providing step comprises displacing the quencher from the linking group.
9. The method of claim 1 , further comprising removing the fragment from the capture element on the surface.
10. The method of claim 9 , wherein the removing step comprises heating the fragment and the capture element.
11. The method of claim 9 , wherein the removing step comprises washing the fragment and the capture element with a buffer.
12. The method of claim 9 , wherein the removing step comprises adding an enzyme to cleave the fragment from the capture element.
13. The method of claim 1 , further comprising cleaving the blocking group from the incorporated nucleotide analogue.
14. The method of claim 1 , wherein the detecting step is performed using fluorescence resonance energy transfer.
15. The method of claim 1 , wherein L is absent.
16. The method of claim 1 , wherein L is a linking group and the linking group is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted aryl.
17. The method of claim 16 , wherein the linking group is a methylene group or a phenyl group.
18. The method of claim 1 , wherein the binding molecule and capture element are a binding pair selected from biotin-streptavidin, biotin-avidin, antibody-antigen, and a complementary oligonucleotide pair capable of forming a nucleic acid duplexes.
19. The method of claim 1 , wherein the blocking group is an amino-containing compound, an allyl-containing compound, an azido-containing compound, an alkoxy-containing compound, polyethylene glycol, or a substituted or unsubstituted alkyl.
20. The method of claim 1 , wherein the blocking group is NH 2 , —CH 2 CH═CH 2 , —CH 2 N 3 , —CH 2 OCH 3 , or polyethylene glycol.