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, a method for determining the nucleic acid make-up of a sample is provided.
1. A method of analyzing a sample nucleic acid, the method comprising:
providing a fluid comprising the sample nucleic acid and a plurality of different primer pairs, wherein each primer pair has at least one unique related probe and is selected to be complementary to one or more sequences of known length;
partitioning the fluid into a plurality of partitions each comprising a maximum of one nucleic acid molecule and more than one of the different primer pairs, wherein at least a first portion of the partitions comprise one molecule of the nucleic acid sample having sequences complementary to one or more of the primer pairs, and at least one related probe, and a second portion of the partitions comprise no molecules of the sample nucleic acid having sequences complementary to one or more of the primer pairs;
conducting a PCR reaction in the partitions, thereby changing a fluorescent property of the first portion of the partitions;
detecting the fluorescent property of each partition;
determining the number of occurrences in the sample nucleic acid of one or more sequences of known length based on the detecting step; and
enriching the sample nucleic acid.
2. The method of claim 1 , further comprising:
comparing a first number of occurrences of a first sequence of a first known length to a second number of occurrences of a second sequence of a second known length.
3. The method of claim 1 , wherein the sample nucleic acid is obtained from a group consisting of blood, saliva, urine, feces, spinal fluid, aspirate, and sputum.
4. The method of claim 1 , wherein the partitions are droplets.
5. The method of claim 4 , wherein the partitioning comprises segmenting the droplets with an immiscible fluid as the aqueous fluid is flowing through a channel.
6. The method of claim 5 , wherein the droplets are surrounded by the immiscible fluid.
7. The method of claim 6 , wherein the immiscible fluid is an oil.
8. The method of claim 7 , wherein the oil comprises a surfactant.
9. The method of claim 8 , wherein the surfactant is a fluorinated surfactant.
10. The method of claim 7 , wherein the oil is a fluorinated oil.
11. The method of claim 10 , wherein the oil is a perfluorocarbon oil.
12. The method of claim 1 , wherein the probe is optically labeled.
13. The method of claim 12 , wherein the probe comprises a fluorescent moiety.
14. The method of claim 1 , further comprising sequencing the sample nucleic acid after enrichment.
15. A method of analyzing a sample nucleic acid, the method comprising:
providing a fluid comprising the sample nucleic acid and a plurality of different primer pairs, wherein each primer pair has at least one unique related probe and is selected to be complementary to one or more sequences of known length;
partitioning the fluid into a plurality of partitions each comprising a maximum of one nucleic acid molecule and more than one of the different primer pairs, wherein at least a first portion of the partitions comprise one molecule of the nucleic acid sample having sequences complementary to one or more of the primer pairs, and at least one related probe, and a second portion of the partitions comprise no molecules of the sample nucleic acid having sequences complementary to one or more of the primer pairs;
conducting a PCR reaction in the partitions, thereby changing a fluorescent property of the first portion of the partitions;
detecting the fluorescent property of each partition;
determining the number of occurrences in the sample nucleic acid of one or more sequences of known length based on the detecting step; and
sequencing the sample nucleic acid.
16. The method of claim 15 , wherein the partitions are droplets.
17. The method of claim 16 , wherein the partitioning comprises segmenting the droplets with an immiscible fluid as the aqueous fluid is flowing through a channel.
18. The method of claim 17 , wherein the droplets are surrounded by the immiscible fluid.
19. The method of claim 15 , wherein the probe is optically labeled.
20. The method of claim 19 , wherein the probe comprises a fluorescent moiety.