Sample multiplexing

• US 8,841,071 B2
• Filed: 05/31/2012
• Issued: 09/23/2014
• Est. Priority Date: 06/02/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Link, Darren
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US13/485,234
• Publication Number:
  US 20120309002A1
• Time in Patent Office: 845 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Abstract

The invention generally relates to methods for sample multiplexing. In certain embodiments, methods of the invention obtaining a plurality of different reactant molecules, attaching a unique identifier to the reactant molecules, and forming a droplet including the reactant molecules.

Claims

1. A method for preparing a plurality of droplets, the method comprising:

obtaining a plurality of reactant molecules from different samples;

attaching a unique identifier to said reactant molecules from each sample so that the reactant molecules from a particular sample can be differentiated from the reactant molecules of other samples; and

forming a plurality of droplets, each droplet comprising reactant molecules from more than one of the different samples and at least one primer pair, wherein a first primer of the primer pair is complementary to a sequence of one of the reactant molecules and a second primer of the primer pair is complementary to a portion of the unique identifier.

2. The method of claim 1, wherein said reactant molecules comprise nucleic acids.

3. The method according to claim 2, wherein the identifier comprises a barcode oligonucleotide portion.

4. The method according to claim 1, further comprising the step of conducting a chemical reaction on said reactant molecules.

5. The method of claim 4, wherein said chemical reaction is a sequencing reaction.

6. The method of claim 4, wherein said chemical reaction is a nucleic acid amplification reaction.

7. The method of claim 6, wherein said amplification reaction is selected from PCR, QPCR, and rolling circle amplification.

8. The method of claim 4, wherein said forming step comprises flowing a stream of sample fluid comprising said reactant molecules, such that the sample fluid stream intersects two opposing streams of flowing carrier fluid, wherein said carrier fluid is immiscible with said sample fluid.

9. The method according to claim 1, wherein said forming step comprises merging at least two droplets, wherein each droplet comprises reactant molecules obtained from different samples.

10. The method according to claim 4, further comprising detecting products of the reaction in the droplet.

11. The method according to claim 2, further comprising releasing the nucleic acids from the droplet.

12. The method of claim 11, further comprising the step of sequencing released nucleic acid.

13. The method according to claim 2, further comprising the steps of:

introducing beads into the droplet; and

attaching the nucleic acids in the droplet to the beads.

14. The method according to claim 13, further comprising releasing the bead-bound nucleic acids from the droplet.

15. The method of claim 14, further comprising sequencing released nucleic acids.

16. The method according to claim 1, wherein subsequent to the forming step, the method further comprises flowing the droplet through a channel.

17. The method according to claim 16, wherein the droplet is surrounded by an immiscible carrier fluid.

18. The method according to claim 17, wherein the immiscible carrier fluid is an oil.

19. The method according to claim 18, wherein the oil comprises a surfactant.

20. The method according to claim 19, wherein the surfactant is a fluorosurfactant.

21. A method for forming a droplet that allows for the differentiation of nucleic acids from different samples, the method comprising:

forming a droplet comprising a primer pair and nucleic acid from different samples, wherein an oligonucleotide sequence comprising a unique sequence portion is attached to the nucleic acids from each sample, so that the nucleic acids from a particular sample can be differentiated from nucleic acids of other samples and a first primer of the primer pair is complementary to a sequence of one of the reactant molecules and a second primer of the primer pair is complementary to a portion of the oligonucleotide sequence.