Field-switch sequencing
The present invention provides novel compositions, methods and apparatus for DNA sequencing that can be performed, e.g., in a two-electrode chamber. The present invention also provides a method for sequencing a nucleic acid comprising immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface, contacting the surface with a plurality of charged particles comprising a nucleotide phosphate by applying an electric field, reversing the electric field to transport unbound charged particles away from the surface, and detecting the incorporation of a nucleotide phosphate into a single molecule of the primer nucleic acid.
1. A method for sequencing a nucleic acid, said method comprising:
(a) immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface;
(b) applying an electric field to contact said surface with a plurality of charged nucleic acid nanoparticles comprising nucleotide phosphate (NP);
(c) removing said nucleotide phosphate (NP) from said charged nucleic acid nanoparticle, wherein said nucleotide phosphate is a substrate for said polymerase;
(d) reversing said electric field to transport unbound charged nucleic acid nanoparticles away from the surface; and
(e) detecting the incorporation of said nucleotide phosphate into a single molecule of the primer nucleic acid.
2. The method according to claim 1 , wherein each of said plurality of charged nucleic acid nanoparticles comprises a plurality of NPs.
3. The method according to claim 1 , wherein said surface comprises a metal, a metal silicide or metal oxide.
4. The method according to claim 3 , wherein said metal or metal oxide is selected from the group consisting of indium, tin, indium-tin, indium-tin oxide, silver, silver oxide, platinum, palladium and gold.
5. The method according to claim 3 , wherein said surface comprises an indium-tin oxide (ITO) electrode.
6. The method according to claim 5 , wherein said ITO electrode is coated by a permeation layer.
7. The method according to claim 6 , wherein said permeation layer is between about 5 nm to about 30 nm thick.
8. The method according to claim 1 , wherein said plurality of charged nucleic acid nanoparticles is selected from the group consisting of a hairpin structure consisting of tandem inverted repeat DNA sequences, a DNA nanoparticle, a RNA nanoparticle, a silica-DNA nanoparticle, a nucleic acid analog nanoparticle, a DNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded DNA, an RNA nanoparticle comprising multiple complementary and non-complementary strands of single stranded RNA, and a combination thereof.
9. The method according to claim 1 , wherein said plurality of charged nucleic acid nanoparticles further comprises at least one dye.
10. The method according to claim 9 , wherein said plurality of charged nucleic acid nanoparticles comprises multiple fluorescent dyes.
11. The method according to claim 1 , wherein detecting comprises detecting a unique signal from the nucleotide phosphate using a system or device selected from the group consisting of an optical reader, a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge couple device, an on-chip multiplication gain charge coupled device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope.
12. The method according to claim 11 , wherein the incorporation of a nucleotide phosphate is detected by a total internal reflection fluorescence microscope.
13. The method according to claim 12 , wherein said total internal reflection fluorescence microscope uses evanescent wave excitation or surface plasmon excitation.
14. The method according to claim 1 , wherein the detecting comprises detecting one or more cleavage molecules.
15. The method according to claim 1 , wherein the detecting comprises detecting said nucleotide phosphate while associated with the polymerase.
16. The method according to claim 1 , wherein said detecting is carried out by a mechanism that enables the detection of single particles.
17. The method according to claim 1 , wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.
18. The method according to claim 1 , wherein polymerase catalyzed nucleotide incorporation is synchronized with the application of said electric field.