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 droplets each comprising a nucleic acid molecule, reagents for an amplification reaction, and a plurality of different optically labeled probes, each specific for a different target sequence;
amplifying a target sequence from the nucleic acid molecule in the droplets;
flowing the droplets past a detector that detects polarized light; and
detecting a level of polarized light emitted from an optically labeled probe that identifies the target sequence in each of the droplets.
2. The method of claim 1 , wherein a high level of polarized light is indicative of an absence of the at least one target sequence.
3. The method of claim 1 , wherein a low level of polarized light is indicative of a positive presence of the at least one target sequence.
4. The method of claim 1 , wherein the optically labeled probes each comprise a species of fluorescent label.
5. The method of claim 1 , wherein the optically labeled probes each comprise a quencher molecule.
6. The method of claim 1 , wherein the optically labeled probes each comprise a moiety that alters polarization of emitted light.
7. The method of claim 1 , wherein the sample is a human tissue or body fluid.
8. The method of claim 1 , wherein a plurality of the droplets each comprise no more than a single nucleic acid molecule.
9. A method for detecting target sequences from a sample, the method comprising:
providing a plurality of droplets each comprising a nucleic acid molecule, reagents for an amplification reaction, and a plurality of different detectably labeled probes, each specific for a different target sequence;
amplifying a target sequence from the nucleic acid molecule in the droplets;
configuring a polarization filter with respect to an excitation polarization;
flowing the droplets past a detector that comprises the polarization filter; and
detecting a level of polarized light emitted from a detectably labeled probe that identifies the target sequence in each of the droplets.
10. The method of claim 9 , wherein the detectably labeled probe comprises a species of fluorescent label.
11. The method of claim 10 , wherein the detector is configured such that the polarization filter is perpendicular to the excitation polarization.
12. The method of claim 9 , wherein a high level of polarized light is indicative of an absence of the at least one target sequence.
13. The method of claim 9 , wherein a low level of polarized light is indicative of a positive presence of the at least one target sequence.
14. The method of claim 9 , wherein the detectably labeled probes each comprise a quencher molecule.
15. The method of claim 9 , wherein the detectably labeled probes each comprise a moiety that alters polarization of emitted light.
16. The method of claim 9 , wherein the sample is a human tissue or body fluid.
17. The method of claim 9 , wherein a plurality of the droplets each comprise no more than a single nucleic acid molecule.
18. The method of claim 10 , wherein the detector is configured such that the polarization filter is parallel to the excitation polarization.
19. The method of claim 1 , wherein the reagents for amplification comprise a plurality of different primer types each specific to amplify a different target sequence.
20. The method of claim 4 , wherein the species of fluorescent label comprises a VIC dye species or a FAM dye species.
21. The method of claim 4 , wherein the species of fluorescent label is released from the optically labeled probes that identifies the target sequence.
22. The method of claim 21 , wherein the level of polarized light detected from the released species of fluorescent label is distinguishable from a level of polarized light emitted from the optically labeled probes that do not identify the target sequence.
23. The method of claim 1 , wherein at least one of the optically labeled probes 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 identify the same target sequence.
24. The method of claim 1 , wherein the different target sequence comprises 5 or fewer different target sequences.