Methods and compositions for nucleic acid analysis

• US 10,190,115 B2
• Filed: 09/22/2014
• Issued: 01/29/2019
• Est. Priority Date: 04/25/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Saxonov, Serge
• Primary Examiner(s): Martinell, James
• Application Number:
  US14/493,272
• Publication Number:
  US 20150011430A1
• Time in Patent Office: 1,590 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Provided herein are methods, compositions, and kits for assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR. The assays may be used for such applications as sequencing, copy number variation analysis, and others. In some cases, the assays involve subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes.

Claims

1. A composition comprising a plurality of second partitions containing first partitions, wherein:

a. said first partitions are degradable upon the application of a stimulus to said first partitions such that contents of a first partition is mixed with contents of a second partition; and

b. said first partitions are contained within the second partitions;

c. said first partitions contain an oligonucleotide barcode; and

d. the first partitions have on average a first average volume and the second partitions have on average a second average volume, wherein the second average volume is at least twice as large as the first average volume.

2. The composition of claim 1, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and combination thereof.

3. The composition of claim 1, wherein said stimulus is a chemical stimulus.

4. The composition of claim 1, wherein said second partition is a droplet.

5. The composition of claim 1, wherein the first partition is a droplet.

6. The composition of claim 1, wherein the first partition and the second partition are droplets.

7. The composition of claim 1, wherein the second partition comprises a sample comprising polynucleotides.

8. The composition of claim 7, wherein the second partition further comprises a DNA polymerase enzyme or a reverse transcriptase enzyme.

9. The composition of claim 7, wherein the polynucleotides comprise DNA.

10. The composition of claim 9, wherein the DNA is genomic DNA.

11. The composition of claim 7, wherein the polynucleotides comprise RNA.

12. The composition of claim 7, wherein the polynucleotides comprise cDNA.

13. The composition of claim 7, wherein the partitions are less than 10 nL in volume.

14. A device comprising a plurality of second partitions, wherein:

a. at least one second partition of the plurality of second partitions contains a first partition comprising an oligonucleotide barcode, and the first partition has a first volume and the at least one second partition has a second volume, wherein the second volume is at least twice as large as the first volume; and

b. said first partition is degradable upon the application of a stimulus to said first partition such that contents of a first partition is mixed with contents of a second partition.

15. The device of claim 14, wherein said second partition is a droplet.

16. The device of claim 14, wherein said stimulus is selected from the group consisting of a biological, chemical, thermal, electrical, magnetic, or photo stimulus, and a combination thereof.

17. The device of claim 16, wherein said stimulus is a chemical stimulus.

18. The device of claim 14, wherein the first partition is a droplet.

19. The device of claim 14, wherein the first partition and the second partition are droplets.

20. The device of claim 19, wherein the droplets are less than 10 nL in volume.

21. The device of claim 14, wherein the second partition comprises a sample comprising polynucleotides.

22. The device of claim 21, wherein the second partition further comprises a DNA polymerase enzyme or a reverse transcriptase enzyme.

23. The device of claim 21, wherein the polynucleotides comprise DNA.

24. The device of claim 23, wherein the DNA is genomic DNA.

25. The device of claim 21, wherein the polynucleotides comprise RNA.

26. The device of claim 21, wherein the polynucleotides comprise cDNA.