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 of performing a digital assay, comprising:
amplifying at least two distinct targets in droplets containing a reporter for each distinct target, wherein the reporters each are labeled with only a same luminophore;
detecting a signal from the droplets representing combined emission of light in a same wavelength from the reporters for the distinct targets, wherein each reporter provides a portion of the signal having an intensity that varies according to whether the corresponding distinct target is present in a droplet, and wherein a plurality of the droplets each contain at least one of the distinct targets and have a distinguishable intensity of the signal relative to droplets containing only one of the distinct targets;
determining for each distinct target a number of droplets that are positive for only one of the distinct targets;
determining at least one number of droplets that are positive for each of the distinct targets; and
determining a quantity representative of a level of at least one of the distinct targets.
2. The method of claim 1 , wherein the quantity representative of a level is a concentration of the at least one distinct target.
3. The method of claim 2 , wherein the step of determining a quantity representative of a level further comprises a step of ascertaining a total number of droplets based on the signal, and obtaining a concentration of the at least one distinct target based on the total number of droplets.
4. The method of claim 1 , wherein the quantity representative of a level is a copy number of the at least one distinct target in a sample disposed in the droplets.
5. The method of claim 1 , wherein the step of determining a quantity representative of a level further comprises a step of obtaining a copy number of a first target by comparing the number of droplets positive for the first target with the number of droplets positive for a second target, the first and second targets being among the at least two distinct targets.
6. The method of claim 5 , wherein the copy number of the second target is known, and wherein the copy number of the first target is determined by calculating the ratio of the number of droplets positive for the first target to the number of droplets positive for the second target and multiplying the ratio by the copy number of the second target.
7. The method of claim 5 , wherein the copy number of the second target is unknown, and wherein the copy number of the first target is obtained relative to the copy number of the second target as the ratio of the number of droplets positive for the first target to the number of droplets positive for the second target.
8. The method of claim 1 , wherein the same luminophore is selected from the group consisting of FAM, VIC, ROX, TAMRA, and JOE.
9. The method of claim 1 , the signal being a first signal, further comprising: detecting from the droplets a second signal representing emission of light from an additional reporter for an additional target, the additional reporter providing at least a portion of a second signal having an intensity that varies according to whether or not the corresponding additional target is present in a droplet, wherein the first signal and second signal correspond to emission of light in a different wavelength; counting a number of the droplets that are positive for the additional target based on the second signal; and determining a quantity representative of a level of the additional target in the droplets.
10. The method of claim 1 , wherein each droplet includes a first probe reporting a presence or absence of a first target in individual droplets, a second probe reporting a presence or absence of a second target in individual droplets, and a third probe reporting a presence or absence of a third target in individual droplets, and wherein a signal from the first and second probes represents light emitted in a first wavelength and a signal from the third probe represents light emitted in a second wavelength distinct from the first wavelength.
11. The method of claim 1 , wherein each reporter is a probe including an oligonucleotide, and wherein each of the distinct targets is a nucleic acid.
12. The method of claim 1 , wherein the intensity of the signal detected from droplets positive for only a first target of the at least two distinct targets is distinguishable from the intensity of the signal detected from droplets positive for only a second target of the at least two distinct targets and wherein the second target has a single base mismatch with at least one reporter.
13. The method of claim 1 , wherein the step of detecting a signal is performed with the droplets disposed in a fluid stream flowing through an examination region.