Digital analyte analysis

• US 9,399,797 B2
• Filed: 04/30/2012
• Issued: 07/26/2016
• Est. Priority Date: 02/12/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Hutchison, John Brian, Olson, Jeffrey Charles, Link, Darren Roy
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/460,762
• Publication Number:
  US 20120302448A1
• Time in Patent Office: 1,548 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

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.

Claims

1. A method for determining a number of nucleic acids that comprise both first and second locations, the method comprising:

partitioning a sample comprising a plurality of nucleic acids of different lengths into partitioned portions, wherein a plurality of the partitioned portions each contain a single nucleic acid;

introducing first and second primer pairs and first and second detectably labeled probes to each of the partitioned portions, wherein the first and second primer pairs are specific for first and second locations on the nucleic acid, the first and second locations being spaced apart from each other, and wherein the first probe hybridizes to the first location and the second probe hybridizes to the second location on the nucleic acid;

amplifying the nucleic acids in the partitioned portions;

detecting amplicons in the partitioned portions, the presence of signal from both probes indicating the presence of a nucleic acid that is contiguous between the first and second locations on the nucleic acid; and

determining the number of nucleic acids that comprise both the first and second locations based upon results of the detecting step.

2. The method of claim 1, wherein the sample is within an aqueous fluid.

3. The method of claim 2, wherein partitioning comprises forming droplets and wherein the partitioned portions are droplets that each contain a single nucleic acid.

4. The method of claim 3, wherein forming comprises segmenting the droplets with an immiscible fluid as the aqueous fluid is flowing through a channel.

5. The method of claim 4, wherein the droplets are surrounded by the immiscible fluid.

6. The method of claim 4, wherein the immiscible fluid is an oil.

7. The method of claim 6, wherein the oil comprises a surfactant.

8. The method of claim 7, wherein the oil comprises a fluorinated surfactant.

9. The method of claim 6, wherein the oil is a fluorinated oil.

10. The method of claim 9, wherein the fluorinated oil is a perfluorocarbon oil.

11. The method of claim 3, wherein introducing comprises forming a droplet comprising the primers and probes.

12. The method of claim 11, comprising merging at least one droplet containing a single nucleic acid with the droplet comprising the primers and probes.

13. The method of claim 12, comprising merging in the presence of an electric field.

14. The method of claim 3, wherein introducing comprises flowing at least one droplet containing a single nucleic acid to interact with a stream of aqueous fluid comprising primers and probes such that a bolus of the aqueous fluid is introduced into the at least one droplet containing the single nucleic acid to form a merged droplet.

15. The method of claim 14, comprising merging in the presence of an electric field.

16. The method of claim 1, wherein the nucleic acid is DNA or RNA.

17. The method of claim 1, wherein the detectably labeled probe comprises an optically labeled probe.

18. The method of claim 17, wherein the optically labeled probe comprises a fluorescent probe.

19. The method of claim 1, wherein the label of the first detectably labeled probe is different from the label of the second detectably labeled probe.

20. The method of claim 1, wherein determining comprises comparing relative amounts of contiguous nucleic acid to relative amounts of non-contiguous nucleic acid.

21. The method of claim 1, wherein determining comprises comparing relative amounts of contiguous nucleic acid or non-contiguous nucleic acid to a total amount of nucleic acid.

22. The method of claim 1, further comprising sequencing the sample after determining the nucleic acid make-up.

23. The method of claim 1, further comprising enriching the sample after determining the number of nucleic acids that comprise both the first and second locations.

24. The method of claim 23, further comprising sequencing the sample after enrichment.

25. The method of claim 1, further comprising fragmenting the sample prior to partitioning.