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 analyzing a nucleic acid, the method comprising:
providing an aqueous fluid in a microchannel, the aqueous fluid comprising nucleic acids, loci-specific primers, and labeled probes labeled with two colors of fluorophores;
intersecting the aqueous fluid with an oil to form a plurality of aqueous droplets, wherein at least one droplet comprises a single target nucleic acid comprising at least three loci, labeled probes for each of the two loci, and a plurality of the loci-specific primers;
conducting an amplification reaction in the droplet; and
detecting via two colors from the labeled probes amplicons produced from the three loci in the droplet to determine a haplotype of the target nucleic acid.
2. The method of claim 1 , wherein the three loci comprise target sequence 1 , target sequence 2 , and target sequence 3 , wherein the probes include: probe P 1 labeled with color 1 that binds to target sequence 1 , probe P 2 labeled with color 2 that binds to target sequence 2 , and probe P 3 labeled with color 1 that binds to target sequence 3 .
3. The method of claim 2 , wherein droplets containing target sequence 1 or 3 emit fluorescence of color 1 at two different intensities and droplets containing target sequence 2 emit fluorescence of color 2 .
4. The method of claim 1 , wherein the nucleic acids are bound to a substrate.
5. The method of claim 1 , wherein the aqueous fluid further comprises a polymerase and a plurality of deoxynucleotides.
6. The method of claim 1 , wherein the nucleic acids and loci-specific primers are combined in the aqueous fluid under microfluidic control.
7. The method of claim 1 , wherein the oil comprises a fluorocarbon oil.
8. The method of claim 1 , wherein the oil comprises a surfactant.
9. The method of claim 1 , further comprising using the haplotype to detect a disease.
10. The method of claim 9 , wherein the disease is cancer, and wherein the loci-specific primers hybridize to loci associated with known cancer biomarkers.
11. The method of claim 1 , further comprising using the haplotype to assess a response to a drug therapy, or identify drug resistance of bacterial or viral infections.
12. The method of claim 1 , wherein the labeled probes for the three loci comprise a probe P 1 labeled with a fluorophore of color 1 that binds to a target 1 and a probe P 2 labeled with a fluorophore of color 2 that binds to a target 2 and a probe P 3 labeled with a fluorophore of color 1 that binds to a target 3 .
13. A method for analyzing a nucleic acid, the method comprising:
providing an aqueous fluid comprising nucleic acids, loci-specific primers, and labeled probes wherein the probes include a probe P 1 labeled with color 1 that binds to target sequence 1 , probe P 2 labeled with color 2 that binds to a target sequence 2 , and probe P 3 labeled with color 1 that binds to a target sequence 3 ;
partitioning the aqueous fluid into aqueous partitions, wherein at least one partition comprises: a single target nucleic acid comprising at least target sequence 1 , target sequence 2 , and target sequence 3 ; the probes; and a plurality of the loci-specific primers;
conducting an amplification reaction in the partition; and
detecting, using two colors from the probes, amplicons produced from target sequence 1 , target sequence 2 , and target sequence 3 in the partition to determine a haplotype of the target nucleic acid.
14. The method of claim 13 , wherein the nucleic acids are obtained from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool, or tissue of an organism.
15. The method of claim 13 , wherein the nucleic acids comprise genomic DNA, cDNA, or RNA.
16. The method of claim 13 , wherein the aqueous fluid further comprises a polymerase and a plurality of deoxynucleotides.
17. The method of claim 13 , further comprising using the haplotype to detect a disease.
18. The method of claim 13 , wherein the disease is cancer, and wherein the loci-specific primers hybridize to loci associated with known cancer biomarkers.
19. The method of claim 13 , further comprising using the haplotype to assess a response to a drug therapy, or identify drug resistance of bacterial or viral infections.