Methods for nucleic acid sequencing
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.
1. A method of identifying a single molecule in a sample comprising two or more types of labeled molecules, the method comprising:
determining a luminescent lifetime and a luminescent intensity of the single molecule in the sample; and
identifying the single molecule as one of the two or more types of labeled molecules based on a combination of both the luminescent lifetime and the luminescent intensity of the single molecule.
2. The method of claim 1 , further comprising delivering a series of pulses of one or more excitation energies to the sample.
3. The method of claim 2 , wherein each of the series of pulses comprises a single excitation energy.
4. The method of claim 2 , wherein a first portion of the series of pulses comprises a first excitation energy and a second portion of the plurality of pulses comprises a second excitation energy.
5. The method of claim 2 , 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.
6. The method of claim 2 , wherein the series of pulses is delivered at an average frequency of between about 50 MHz and about 200 MHz.
7. The method of claim 1 , further comprising detecting a plurality of emitted photons from the sample.
8. The method of claim 7 , 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.
9. The method of claim 1 , 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.
10. The method of claim 1 , wherein the two or more types of labeled molecules comprise four types of molecules.
11. The method of claim 10 , wherein the four types of molecules comprise four types of luminescently labeled nucleotides.
12. The method of claim 11 , 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.
13. The method of claim 1 , wherein the single molecule is a luminescently labeled nucleotide incorporated into a nucleic acid.
14. The method of claim 1 , wherein the method is repeated to identify a series of single molecules.
15. The method of claim 14 , wherein the series of single molecules is a series of luminescently labeled nucleotides sequentially incorporated into a nucleic acid comprising a primer.
16. The method of claim 15 , 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.
17. The method of claim 1 , wherein each single molecule identified is immobilized on a solid support.
18. The method of claim 17 , wherein the solid support comprises a bottom surface of a sample well.
19. The method of claim 1 , wherein the luminescent lifetime is determined by measuring a distribution of emission events over time, and wherein the luminescent intensity is determined by measuring an amount of emission events across the distribution.