Single molecule sequencing of captured nucleic acids
View Patent ↗The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.
1. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:
introducing a sample comprising at least one target nucleic acid and other non-target molecules to at least one sequence-specific capture probe to produce target/probe duplexes;
separating at least one of the duplexes from the non-target molecules;
exposing the separated duplexes to at least one labeled nucleotide, wherein the label is attached to a nitrogenous base of the labeled nucleotide, in the presence of a polymerase having reduced exonuclease activity capable of catalyzing addition of the nucleotide to the duplex in a template-dependent manner, wherein the probe acts as a primer; and
detecting incorporation of the nucleotide into the probe portion, thereby obtaining sequence information from the nucleic acid.
2. The method according to claim 1 , wherein prior to the exposing step, the duplexes are attached to a surface.
3. The method according to claim 2 , wherein the duplexes are directly attached to a surface.
4. The method according to claim 2 , wherein the duplexes are indirectly attached to a surface.
5. The method according to claim 2 , wherein at least some of the attached duplexes are individually optically resolvable.
6. The method according to claim 1 , wherein the probes are a plurality of different sequence-specific probes.
7. The method according to claim 1 , wherein the exposing and detecting steps are repeated at least once.
8. The method according to claim 7 , further comprising:
removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.
9. The method according to claim 1 , wherein the labeled nucleotide is a detectably labeled nucleotide.
10. The method according to claim 9 , wherein the detectably labeled nucleotide is an optically detectably labeled nucleotide.
11. The method according to claim 10 , wherein the optically detectably labeled nucleotide is a fluorescently labeled nucleotide.
12. The method according to claim 1 , wherein the target nucleic acid comprises at least one mutation indicative of a disease state.
13. The method according to claim 1 , wherein prior to the introducing step, the method further comprising modifying the target nucleic acid with an adaptor sequence.
14. The method according to claim 13 , wherein each target nucleic acid is modified with a different adaptor sequence.
15. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:
contacting a sample comprising at least one target nucleic acid and other non-target molecules to at least one sequence-specific capture probe attached to a surface to produce target/probe duplexes;
separating at least one of the duplexes from the non-target molecules;
exposing the separated duplexes to at least one labeled nucleotide, wherein the label is attached to a nitrogenous base of the labeled nucleotide, in the presence of a polymerase having reduced exonuclease activity capable of catalyzing addition of the nucleotide to the duplex in a template-dependent manner, wherein the probe acts as a primer; and
detecting incorporation of the nucleotide into the probe portion, thereby obtaining sequence information from the nucleic acid.
16. The method according to claim 15 , wherein at least some of the attached duplexes are individually optically resolvable.
17. The method according to claim 15 , wherein the exposing and detecting steps are repeated at least once.
18. The method according to claim 17 , further comprising: removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.
19. The method according to claim 15 , wherein the probes are directly attached to the surface.
20. The method according to claim 15 , wherein the probes are indirectly attached to the surface.
21. The method according to claim 15 , wherein the probes are a plurality of different sequence-specific probes.
22. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:
performing hybrid capture on a sample comprising at least one target nucleic acid and other non-target molecules to produce target/probe duplexes;
separating at least one of the duplexes from the non-target molecules; and performing a sequencing by synthesis reaction on the separated target/probe
duplexes in the presence of a polymerase having reduced exonuclease activity and at least one labeled nucleotide, wherein the label is attached to a nitrogenous base of the labeled nucleotide, and wherein the probe acts as a primer for the sequencing reaction.