METHODS FOR NUCLEIC ACID SEQUENCING

• US 20170107562A1
• Filed: 05/20/2016
• Published: 04/20/2017
• Est. Priority Date: 05/20/2015
• Status: Active Grant

• Current Assignee: Quantum-Si Incorporated
• Original Assignee: Quantum-Si Incorporated
• Inventors: Reed, Brian, Shi, Xinghua, Huang, Haidong, Rothberg, Jonathan M., Lackey, Jeremy
• Granted Patent: US 10,174,363 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6454 using an integrated detector array
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 2201/0697 Pulsed lasers

Abstract

Methods of sequencing molecules based on luminescence lifetimes and/or intensities are provided. In some aspects, methods of sequencing nucleic acids involve determining the luminescence lifetimes, and optionally luminescence intensities, of a series of luminescently labeled nucleotides incorporated during a nucleic acid sequencing reaction.

Claims

1. A method of identifying a single molecule in a sample, the method comprising determining a luminescent lifetime and optionally a luminescent intensity of the single molecule.

2. The method of claim 1, further comprising determining both the luminescent lifetime and the luminescent intensity of the single molecule.

3. The method of any one of the preceding claims, further comprising delivering a series of pulses of one or more excitation energies to the sample.

4. The method of any one of the preceding claims, further comprising detecting a plurality of emitted photons from the sample.

5. The method any one of the preceding claims, further comprising recording for each of a plurality of emitted photons from the sample a time duration between detection of the emitted photon and a prior delivered excitation energy.

6. The method of any one of the preceding claims, wherein identifying comprises identifying the single molecule as one type out of a plurality of possible types of molecules.

7. The method of claim 6, wherein the plurality of possible types of molecules comprises four types of molecules.

8. The method of claim 7, wherein the four types of molecules comprise four types of luminescently labeled nucleotides.

9. The method of claim 8, wherein the four types of luminescently labeled nucleotides comprises a first nucleotide comprising adenine, a second nucleotide comprising guanine, a third nucleotide comprising cytosine, and a fourth nucleotide comprising thymine or uracil.

10. The method of claim 8, wherein the single molecule is a luminescently labeled nucleotide incorporated into a nucleic acid.

11. The method of any of the preceding claims, wherein the method is repeated to identify a series of single molecules.

12. The method of claim 11, wherein the series of single molecules is a series of luminescently labeled nucleotides sequentially incorporated into a nucleic acid comprising a primer.

13. The method of claim 12, further comprising determining the sequence of a template nucleic acid by analyzing the sequence of luminescently labeled nucleotides incorporated sequentially into the nucleic acid comprising the primer.

14. The method of any one of the preceding claims, wherein each of the plurality of pulses comprises a single excitation energy.

15. The method of any one of the preceding claims, wherein a first portion of the plurality of pulses comprises a first excitation energy and a second portion of the plurality of pulses comprises a second excitation energy.

16. The method of any one of the preceding claims, wherein the series of pulses is delivered at an average frequency of between about 10 MHz and about 100 MHz or between about 100 MHz and about 1 GHz.

17. The method of claim 16, wherein the frequency is between about 50 MHz and about 200 MHz.

18. The method of any one of the preceding claims, wherein for each single molecule identified, the number of detected emitted photons is between about 10 and about 100, or between about 100 and about 1,000.

19. The method of any one of the preceding claims, wherein each single molecule identified is immobilized on a solid support.

20. The method of claim 19, wherein the solid support comprises a bottom surface of a sample well.

21. A method of determining the sequence of a template nucleic acid comprising:

(i) exposing a complex in a target volume, the complex comprising the template nucleic acid, a primer, and a polymerizing enzyme, to a plurality of types of luminescently labeled nucleotides, wherein each type of luminescently labeled nucleotide has a different luminescent lifetime and/or luminescent intensity;

(ii) directing a series of pulses of one or more excitation energies towards a vicinity of the target volume;

(iii) detecting a plurality of emitted photons from luminescently labeled nucleotides during sequential incorporation into a nucleic acid comprising the primer; and

(iv) identifying the sequence of incorporated nucleotides by determining timing and optionally frequency of the emitted photons.

22. The method of claim 21, wherein detecting further comprises recording for each detected emitted photon a time duration between detection of the emitted photon and a prior pulse of excitation energy.

23. The method of claim 21 or 22, wherein each type of luminescently labeled nucleotide emits photons after being illuminated by a single excitation energy.

24. The method of claim 23, wherein the single excitation energy is in a spectral range of about 470 to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm.

25. The method of claim 21, wherein one or more type of luminescently labeled nucleotide emits photons after being illuminated by a first excitation energy, and one or more type of luminescently labeled nucleotide emits photons after being illuminated by a second excitation energy.

26. The method of claim 25, wherein the first excitation energy is in a spectral range of about 470 nm to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm, and the second excitation energy is in a spectral range of about 470 to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm.

27. The method of claim 26, wherein the first excitation energy is in a spectral range of about 520 to about 550 nm, and the second excitation energy is in a spectral range of about 550 to about 580 nm.

28. The method of claim 26, wherein the first excitation energy is in a spectral range of about 520 to about 550 nm, and the second excitation energy is in a spectral range of about 580 to about 620 nm.

29. The method of claim 26, wherein the first excitation energy is in a spectral range of about 520 to about 550 nm, and the second excitation energy is in a spectral range of about 560 to about 670 nm.

30. The method of claim 26, wherein the first excitation energy is in a spectral range of about 550 to about 580 nm, and the second excitation energy is in a spectral range of about 580 to about 620 nm.

31-86. -86. (canceled)