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 microdroplet comprising a single nucleic acid template and a plurality of primer pairs, each primer pair specific for a different nucleic acid template, wherein a member of each primer pair comprises first and second targeting arms coupled via a bridge section, the first and second targeting arms each comprising a sequence that hybridizes to a nucleic acid template and the bridge section comprising a sequence that does not hybridize to the nucleic acid template.
2. The microdroplet of claim 1 , further comprising a plurality of probes, wherein each probe hybridizes to a complement of the bridge section of a primer pair member.
3. The microdroplet of claim 1 , wherein the single nucleic acid template is a DNA or an RNA.
4. The microdroplet of claim 1 , further comprising reagents for conducting an amplification reaction.
5. The microdroplet of claim 4 , further comprising reagents for conducting a polymerase chain reaction.
6. The microdroplet of claim 2 , wherein each probe comprises a detectable label.
7. The microdroplet of claim 6 , wherein the probes belong to one or more groups, wherein each group is at a different concentration.
8. The microdroplet of claim 7 , wherein members of each group of probes comprise the same detectable label.
9. The microdroplet of claim 6 , wherein members of each group of probes comprise a different detectable label.
10. The microdroplet of claim 6 , wherein the detectable label is a fluorescent label.
11. The microdroplet of claim 1 , wherein the primer pairs are specific for nucleic acid templates from different chromosomes.
12. A method for detecting a target in a biological sample, said method comprising:
providing one or more droplets each comprising: a single nucleic acid template, a plurality of primer pairs, and a plurality of probes, wherein each primer pair is specific for a different nucleic acid template; a member of each primer pair comprises first and second targeting arms coupled via a bridge section, the first and second targeting arms each comprising a sequence that hybridizes to the nucleic acid template and the bridge section comprising a sequence that does not hybridize to the nucleic acid template; and each probe hybridizes to a complement of the bridge section of a primer pair member;
amplifying the nucleic acid template within the droplet; and
detecting a signal from a label of a probe that hybridizes to a complement of the bridge section of an amplified product, thereby detecting the target.
13. The method of claim 12 , further comprising generating one or more droplets by contacting:
a first fluid comprising a single nucleic acid template;
with a second fluid comprising a plurality of primer pairs and a plurality of probes, wherein each primer pair is specific for a different nucleic acid template; a member of each primer pair comprises first and second targeting arms coupled via a bridge section, the first and second targeting arms each comprising a sequence that hybridizes to the nucleic acid template; and each probe hybridizes to a complement of the bridge section of a primer pair member.
14. The method of claim 13 , wherein the first and second fluids are contacted in the presence of an electric field.
15. The method of claim 13 , wherein the second fluid further comprises reagents for conducting an amplification reaction.
16. The method of claim 13 , wherein a droplet comprising said first fluid is contacted with a droplet comprising the second fluid.
17. The method of claim 15 , wherein the second fluid further comprises reagents for conducting a polymerase chain reaction.
18. The method of claim 13 , wherein the single nucleic acid template is a DNA or an RNA.
19. The method of claim 13 , wherein the probes belong to one or more groups, wherein each group is at a different concentration.
20. The method of claim 19 , wherein members of each group of probes comprise the same detectable label.
21. The method of claim 19 , wherein members of each group of probes comprise a different detectable label.
22. The method of claim 13 , wherein the detectable label is a fluorescent.
23. The method of claim 13 , wherein the plurality of primer pairs are specific to nucleic acid templates from different chromosomes.
24. The microdroplet of claim 1 , further comprising a plurality of second primer pairs, each primer pair specific for a different target site on a nucleic acid template.
25. The method of claim 12 , wherein the one or more droplets each further comprising a plurality of second primer pairs, each primer pair specific for a different target site on a nucleic acid template.