Digital analyte analysis
View Patent ↗The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.
1. A method for detecting target sequences from a sample, the method comprising:
providing a plurality of partitions each comprising a nucleic acid molecule, reagents for an amplification reaction comprising a plurality of different primer types each specific to amplify a different target sequence, and a plurality of optically labeled probe types each specific for a different target sequence;
amplifying a single target sequence from the nucleic acid molecule in each droplet; and
detecting light emitted from an optically labeled probe type specific to the amplified target sequence in the partitions.
2. The method of claim 1 , further comprising,
identifying the amplified target sequence in the droplets using a combination of a wavelength and an intensity of the detected light.
3. The method of claim 2 , wherein the step of identifying comprises identifying one or more of the droplets that do not comprise an amplified target sequence.
4. The method of claim 2 , wherein the step of identifying comprises identifying up to 9 amplified target sequences in the droplets using a combination of a first wavelength, a second wavelength, and the intensity of the detected light.
5. The method of claim 2 , wherein the step of identifying comprises expression analysis of the amplified target sequence.
6. The method of claim 2 , wherein the nucleic acid molecules are from a sample, a first optically labeled probe type is specific for a reference target sequence, a second optically labeled probe type is specific for a copy number variant target sequence; and the step of identifying comprises determining a number of the identified droplets comprising the reference target sequence and a number of the identified droplets comprising the copy number variant target sequence, wherein a ratio of the number of reference droplets to the number of copy number variant droplets determines a gene copy number of the copy number variant sequence.
7. The method of claim 6 , wherein the sample is from a patient and the copy number variant sequence comprises SMN1 or SMN2.
8. The method of claim 1 , wherein the optically labeled probe types each comprise a species of fluorescent label.
9. The method of claim 8 , wherein the optically labeled probe types comprise hydrolysis probes and the species of fluorescent label comprises a VIC dye species or a FAM dye species.
10. The method of claim 1 , wherein the sample is a human tissue or body fluid.
11. The method of claim 1 , wherein a plurality of the droplets each comprise no more than a single nucleic acid molecule.
12. The method of claim 1 , wherein at least one of the optically labeled probe types specific for a target sequence comprises a first sub-type comprising a first fluorescent label and a second sub-type comprising a second fluorescent label different than the first, wherein the first and second sub-types are specific to the same target sequence.
13. The method of claim 1 , wherein at least one of the optically labeled probe types specific for a target sequence comprises a first sub-type comprising a first fluorescent label and a second sub-type without a fluorescent label, wherein the first and second sub-types are specific to the same target sequence.
14. The method of claim 9 , wherein the droplets comprise droplets.
15. A method for detecting target sequences from a sample, the method comprising:
providing a plurality of droplets each comprising reagents for an amplification reaction comprising a plurality of different primer types each specific to amplify a different target sequence, and a plurality of different optically labeled probe types each specific for a different target sequence;
introducing a nucleic acid molecule into a plurality of the droplets;
amplifying a single target sequence from the nucleic acid molecule in the droplets; and
detecting light emitted from an optically labeled probe type specific to the amplified target sequence in the droplets.
16. The method of claim 15 , wherein the step of introducing comprises fusing each of the droplets comprising the reagents for an amplification reaction, and the plurality of different optically labeled probe types with a second droplet that comprises the nucleic acid molecule.
17. The method of claim 15 , wherein the step of introducing comprises integrating a portion of a fluid stream comprising the nucleic acid molecule with each of the droplets.
18. The method of claim 15 , further comprising,
identifying the amplified target sequence in droplets using a combination of a wavelength and an intensity of the detected light.
19. The method of claim 18 , wherein the step of identifying comprises identifying one or more of the droplets that do not comprise an amplified target sequence.
20. The method of claim 18 , wherein the step of identifying comprises identifying up to 9 amplified target sequences in the droplets using a combination of a first wavelength, a second wavelength, and the intensity of the detected light.
21. The method of claim 15 , wherein the optically labeled probe types each comprise a species of fluorescent label.
22. The method of claim 21 , wherein the optically labeled probe types comprise hydrolysis probes and the species of fluorescent label comprises a VIC dye species or a FAM dye species.
23. The method of claim 1 , wherein the reagents for amplification comprise a polymerase species and a plurality of deoxynucleotide species into the droplets.
24. The method of claim 1 , wherein the step of introducing further comprises introducing a polymerase species and a plurality of deoxynucleotide species into the droplets.
25. The method of claim 15 , wherein at least one of the optically labeled probe types specific for a target sequence comprises a first sub-type comprising a first fluorescent label and a second sub-type comprising a second fluorescent label different than the first, wherein the first and second sub-types are specific to the same target sequence.
26. The method of claim 15 , wherein at least one of the optically labeled probe types specific for a target sequence comprises a first sub-type comprising a first fluorescent label and a second sub-type without a fluorescent label, wherein the first and second sub-types are specific to the same target sequence.
27. The method of claim 15 , wherein the step of providing further comprises:
providing one or more optical identifier labels in the droplets and the reagents for amplification comprise a plurality of different primer types each specific to amplify a different target sequence, wherein each of the optical identifier labels is specific to a different assay; and
the step of detecting further comprises detecting light emitted from the optical identifier labels.
28. The method of claim 15 , wherein the optical identifier labels comprise one or more fluorophores each of which is spectrally distinct from the optically labeled probe types.