Claims
- 1. A method of distinguishing between a plurality of fluorescent species, wherein each of said fluorescent species has a fluorescence emission, said emission having a characteristic fluorescence lifetime, said method comprising:
(a) electrokinetically transporting each of said species through a microfluidic channel; (b) detecting each of said fluorescent species in said channel; and (c) identifying each of said fluorescent species by measuring said characteristic fluorescence lifetime.
- 2. The method according to claim 1, wherein said microfluidic channel is in a microfluidic apparatus comprising two or more intersecting channels.
- 3. The method according to claim 2, wherein said microfluidic apparatus comprises:
a glass or polymer body having at least two intersecting channels fabricated therein, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range of from about 0.1 μm to about 500 μm.
- 4. A method of sequencing a nucleic acid polymer of interest, said method comprising:
(a) performing a sequencing reaction on said nucleic acid polymer utilizing a sequencing reaction mixture comprising said nucleic acid polymer and a first labeled nucleic acid bearing a first fluorescent label, wherein said fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime; (b) electrokinetically transporting each of said species through a microfluidic channel; (c) detecting said first labeled nucleic acid bearing a first fluorescent label; and (d) identifying said first labeled nucleic acid bearing a first fluorescent label by measuring said characteristic fluorescence lifetime.
- 5. The method according to claim 4, wherein said sequencing reaction mixture further comprises a second labeled nucleic acid bearing a second fluorescent label, wherein said second fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 6. The method according to claim 5, wherein said sequencing reaction mixture further comprises a third labeled nucleic acid bearing a third fluorescent label, wherein said third fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 7. The method according to claim 6, wherein said sequencing reaction mixture further comprises a fourth labeled nucleic acid bearing a fourth fluorescent label, wherein said fourth fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 8. The method according to claim 4, wherein said first labeled nucleic acid bearing a first fluorescent label is a member of a plurality of unique labeled nucleic acid species, wherein each unique labeled nucleic acid species comprises a unique fluorescent label having a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 9. The method according to claim 4 further comprising a second sequencing reaction mixture comprising said nucleic acid polymer of interest and a second labeled nucleic acid bearing a second fluorescent label, wherein said second fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 10. The method according to claim 9 further comprising a third sequencing reaction mixture comprising said nucleic acid polymer of interest and a third labeled nucleic acid bearing a third fluorescent label, wherein said third fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 11. The method according to claim 10 further comprising a fourth sequencing reaction mixture comprising said nucleic acid polymer of interest and a fourth labeled nucleic acid bearing a fourth fluorescent label, wherein said fourth fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime.
- 12. The method according to claim 9, wherein said first sequencing reaction mixture and said second sequencing reaction mixture are members of a plurality of unique sequencing reaction mixtures, each reaction mixture comprising said nucleic acid polymer of interest and a unique labeled nucleic acid bearing a unique fluorescent label, wherein said unique fluorescent label has a fluorescence emission, said emission having a characteristic fluorescence lifetime, said lifetime being different for each unique fluorescent label.
- 13. The method according to claim 4, wherein said sequencing reaction mixture further comprises one or more members selected from the group consisting of polymerases, exonucleases, endonucleases, deoxynucleotides, deoxynucleotide diphosphates, deoxynucleotide triphosphates, dideoxynucleotides, dideoxynucleotide diphosphates, dideoxynucleotide triphosphates, nucleotide analogs and nucleoside analogs and combinations thereof.
- 14. The method according to claim 4, wherein the labeled nucleic acids are members selected from the group consisting of nucleotides, nucleosides, nucleoside diphosphates, nucleoside triphosphates, dideoxynucleosides, deoxynucleotides, deoxynucleoside diphosphates, deoxynucleoside triphosphates, dideoxynucleosides, dideoxynucleotides, dideoxynucleoside diphosphates, dideoxynucleoside triphosphates, nucleotide analogs and nucleoside analogs and combinations thereof.
- 15. The method according to claim 4, wherein said nucleotide bearing a fluorescent label is a non-natural nucleotide.
- 16. The method according to claim 4, wherein said nucleotide bearing a fluorescent label comprises a base segment and a sugar segment and said fluorescent label is covalently attached to a segment which is a member selected from the group consisting of said base segment, said sugar segment and both said base segment and said sugar segment.
- 17. The method according to claim 4 further comprising, prior to step (b), producing a plurality of nucleic acid polymers complementary to a region of said nucleic acid polymer of interest.
- 18. The method according to claim 17 further comprising separating said complementary nucleic acid polymers into distinct populations, each of said populations consisting of nucleic acid polymers of about the same size.
- 19. The method according to claim 4, wherein said first fluorescent label is not a lanthanide chelate.
- 20. The method according to claim 17, wherein said nucleic acid polymer of interest is of a size of from about 2 bases to about 100,000 bases.
- 21. The method according to claim 20, wherein said nucleic acid polymer of interest is of a size of from about 100 bases to about 10,000 bases.
- 22. The method according to claim 21, wherein said nucleic acid polymer of interest is of a size of from about 300 bases to about 5000 bases.
- 23. The method according to claim 4 further comprising annealing said nucleic acid polymer of interest with a primer nucleic acid polymer.
- 24. The method according to claim 23, wherein said primer nucleic acid polymer comprises at least one labeled nucleic acid bearing a fluorescent label.
- 25. The method according to claim 24, wherein said fluorescent label is covalently attached to a labeled nucleic acid which is a member selected from the group consisting of the 3′-terminus, the 5′-terminus, an internal position and combinations thereof.
- 26. The method according to claim 5, wherein said first fluorescent label and said second fluorescent label have an emission maximum which occurs at substantially the same wavelength.
- 27. The method according to claim 5, wherein said first fluorescent label and said second fluorescent label have an emission maximum which occurs at a substantially different wavelength.
- 28. The method according to claim 4, wherein said fluorescent label has a fluorescence lifetime of from about 1 nanosecond to about 4000 nanoseconds.
- 29. The method according to claim 28, wherein said fluorescent label has a fluorescence lifetime of from about 1 nanosecond to about 1000 nanoseconds.
- 30. The method according to claim 29, wherein said fluorescent label has a fluorescence lifetime of from about 1 nanoseconds to about 100 nanoseconds.
- 31. The method according to claim 4, wherein said detecting is provided by a pulse method or a phase-modulation method.
- 32. The method according to claim 4, wherein said sequencing is performed using a microfluidic apparatus.
- 33. The method according to claim 32, wherein said detecting is performed using a microfluidic apparatus.
- 34. The method according to claim 32, wherein said identifying by measuring is performed using a microfluidic apparatus.
- 35. The method according to claim 32, wherein said microfluidic apparatus comprises:
a glass or polymer body having therein, at least two intersecting channels fabricated into said surface of said substrate, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range of from about 0.1 μm to about 500 μm.
- 36. A method according to claim 4 further comprising the polymerase chain reaction.
- 37. An apparatus for distinguishing between a plurality of fluorescent species, wherein each of said fluorescent species has a fluorescence emission, said emission having a characteristic fluorescence lifetime comprising:
a microfluidic device with at least one microchannel therein; a detector for detecting fluorescence emissions by the fluorescent species in the at least one channel; a digital computer operably linked to the detector for determining fluorescence lifetimes of the fluorescent species.
- 38. The apparatus according to claim 37, wherein said microfluidic apparatus comprises:
a glass or polymer body having at least two intersecting channels fabricated therein, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range of from about 0.1 μm to about 500 μm.
- 39. A sequencing reaction mixture comprising:
a first fluorescent label having a first fluorescent lifetime, a second fluorescent label having a second fluorescent lifetime, wherein said first fluorescent lifetime and said second fluorescent lifetime are different; and a first nucleic acid and a second nucleic acid, said first nucleic acid and said second nucleic acid being members independently selected from the group consisting of nucleotides, nucleosides, nucleoside diphosphates, nucleoside triphosphates, dideoxynucleosides, deoxynucleotides, deoxynucleoside diphosphates, deoxynucleoside triphosphates, dideoxynucleosides, dideoxynucleotides, dideoxynucleoside diphosphates, dideoxynucleoside triphosphates, nucleotide analogs and nucleoside analogs.
- 40. A kit comprising a sequencing mixture according to claim 39.
- 41. A kit comprising a sequencing mixture according to claim 39 and a microfluidic device.
- 42. A method of distinguishing between a plurality of fluorescent species, wherein each of said fluorescent species has a fluorescence emission, said emission having a characteristic fluorescence lifetime;
(a) electrokinetically separating each of said species; (b) detecting each of said fluorescent species; and (c) identifying each of said fluorescent species by measuring said characteristic fluorescence lifetime.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of U.S. patent application Ser. No. 09/132,181, filed Aug. 11, 1998 entitled “DNA Sequencing Using Multiple Fluorescent Labels Being Distinguishable by Their Decay Times,” and is related to U.S. patent application Ser. No. 09/132,554, filed Aug. 11, 1998, entitled “Methods and Systems for Sequencing DNA by Distinguishing the Decay Times of Fluorescent Probes,” the disclosures of both of which are incorporated herein by reference in their entirety for all purposes.
Provisional Applications (1)
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Number |
Date |
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60122064 |
Aug 1998 |
US |
Continuations (1)
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Number |
Date |
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Parent |
09213297 |
Dec 1998 |
US |
Child |
10179686 |
Jun 2002 |
US |
Continuation in Parts (1)
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Number |
Date |
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Parent |
09132181 |
Aug 1998 |
US |
Child |
09213297 |
Dec 1998 |
US |