Digital analyte analysis
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 nucleic acid sequences in a sample, the method comprising:
(a) partitioning a sample comprising a plurality of nucleic acid molecules comprising one or more target nucleic acid sequences into compartments, wherein a plurality of the compartments each comprise one target nucleic acid sequence, amplification reagents comprising a plurality of primer pairs, and a plurality of detectably labeled probes, and wherein each primer pair is specific to amplify a different target nucleic acid sequence and each detectably labeled probe is specific for a different target nucleic acid sequence, wherein the plurality of detectably labeled probes include:
a first probe specific for a first target of the one or more target nucleic acid sequences, wherein the first probe includes a first fluorophore and is present at a first concentration, and
a second probe specific for a second target of the one or more target nucleic acid sequences, wherein the second probe includes the first fluorophore and is present at a second concentration different from the first concentration;
(b) amplifying target nucleic acid sequences in one or more of the compartments to generate fluorescence at a first fluorescent intensity from compartments that contain the first target due to the first concentration of the first probe and to generate the fluorescence at a second fluorescent intensity, different from the first fluorescent intensity, from compartments that contain the second target due to the second concentration of the second probe; and
(c) detecting the first fluorescent intensity and the second fluorescent intensity to detect presence or absence of each of the first and second targets in the sample.
2. The method of claim 1 , wherein the first fluorophore is selected from the group consisting of coumarin, FAM, VIC, ROX, an Atto dye, a fluorescein derivative, HEX, Texas Red, Cy5, and Cy5.5.
3. The method of claim 1 , wherein the detecting step creates a 2D plot of fluorescence intensities from the first fluorophore and a second fluorophore from the detectably labeled probes.
4. The method of claim 3 , wherein one or both of the first and the second fluorophore are selected from the group consisting of FAM, VIC, ROX, an Atto dye, a fluorescein derivative, coumarin, Texas Red, HEX, Cy5, and Cy5.5.
5. The method of claim 3 , wherein each of the target nucleic acid sequences forms a distinct cluster on the 2D plot.
6. The method of claim 1 , wherein the sample is partitioned into the compartments at limiting dilution.
7. The method of claim 1 , wherein the amplifying and detecting steps comprise digital PCR.
8. The method of claim 1 , wherein the compartments are microwells.
9. The method of claim 1 , wherein the detecting step is performed using a microscope, photomultiplier tube, a CCD camera, or a photodiode.
10. The method of claim 1 , wherein at least two fluorophores from the detectably labeled probes are used for multiplex detection of the target nucleic acid sequences.
11. The method of claim 1 , wherein the compartments are not droplets.
12. The method of claim 1 , wherein the compartments are containers.
13. The method of claim 1 , wherein one or more of the detectably labeled probes are labeled with a fluorophore selected from the group consisting of FAM, VIC, ROX, an Atto dye, a fluorescein derivative, coumarin, Texas Red, HEX, Cy5, and Cy5.5.