COMPOSITIONS AND METHODS FOR MOLECULAR LABELING

• US 20120220494A1
• Filed: 02/16/2012
• Published: 08/30/2012
• Est. Priority Date: 02/18/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Samuels, Michael, Link, Darren Roy, Olson, Jeffrey Charles, Watson, Andrew, Brown, Keith
• Granted Patent: US 9,150,852 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/10 Nucleotidyl transfering
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

Claims

1. A method of making a barcode library comprising:

obtaining a plurality of nucleic acid constructs wherein each construct comprises a unique N-mer and a functional N-mer; and

segregating the plurality of constructs into fluid compartments such that each fluid compartment contains one or more copies of a unique construct.

2. The method of claim 1, wherein the functional N-mer is a random N-mer.

3. The method of claim 1, wherein the functional N-mer is configured to operate as a PCR primer.

4. The method of claim 1, wherein the functional N-mer is configured to operate as a universal primer.

5. The method of claim 1, wherein the functional N-mer is a sticky end.

6. The method of claim 1, where in the fluid compartments are water-in-oil droplets.

7. The method of claim 1, further comprising:

(a) preparing N of the fluid compartments containing the unique constructs;

(b) adding an additional construct to each fluid compartment; and

(c) repeating steps (a) and (b) M times, thereby producing M×

N fluid compartments each containing a unique pair of constructs.

8. The method of claim 7, wherein adding an additional construct to a fluid compartment comprises merging droplets in a microfluidic device.

9. The method of claim 7, further comprising ligating the unique pairs of constructs to form new constructs.

10. The method of claim 1, wherein the unique N-mer is adapted for identification by sequencing.

11. The method of claim 1, wherein the unique N-mer is adapted for identification by digital PCR.

12. The method of claim 1 further comprising:

hybridizing a second construct to each of the plurality of constructs to form a hybrid construct;

ligating the hybrid constructs to form new constructs.

13. The method of claim 1, wherein each of the unique N-mers is between 3 and 30 nucleotides in length.

14. The method of claim 1, wherein the functional N-mer comprises an antibody.

15. A method for labeling target material comprising:

segregating each of a plurality of targets into a fluid compartment;

providing one or more copies of a construct that is unique for each fluid compartment, wherein each construct includes a unique N-mer and a functional N-mer; and

associating each target with copies of the construct unique to the fluid partition.

16. The method of claim 15 wherein the target comprises one selected from the list consisting of:

a cell;

a protein;

a construct comprising a unique N-mer and a functional N-mer;

a genome;

a complex of molecules; and

a nucleic acid.

17. The method of claim 15, wherein each functional N-mer comprises one selected from the list consisting of:

sticky end;

universal primer site;

sequence-specific primer;

antibody;

probe target; and

random N-mer.

18. The method of claim 15, further comprising:

hybridizing each functional N-mer to a target nucleic acid strand;

copying the target nucleic strand to form a product that includes the unique N-mer;

releasing contents from the fluid compartments and allowing the contents to mix.

19. The method of claim 15, further comprising:

(a) performing an amplification reaction to produce amplicons that each contain a copy of the construct; and

(b) releasing the contents of the fluid partitions into a bulk phase, wherein the order of the steps includes one selected from the list consisting of;

(a) then (b);

(b) then (a); and

(a) while (b).

20. The method of claim 19, further comprising performing a second amplification reaction on the amplicons.

21. The method of claim 19, further comprising sequencing the amplicons.

22. The method of claim 15, further comprising:

introducing into each fluid partition one or more additional constructs that are unique to portions of the target; and

linking each additional construct to a copy of the construct unique to the fluid partition.

23. The method of claim 15, wherein the targets are unlabeled when segregated into the fluid compartments.

24. The method of claim 15, wherein each target comprises a strand of nucleic acid, and further wherein the method comprises:

hybridizing a copy of the construct to the strand; and

copying at least a portion of the strand using the hybridized construct as a primer.

25. The method of claim 24, wherein the copying is done by T4 polymerase.

26. The method of claim 15, wherein the targets are cells, and further wherein the method comprises:

lysing the cells within the fluid compartments; and

hybridizing the constructs to lysate from the cells within the fluid compartments.

27. The method of claim 15, wherein the functional N-mer comprises a capture tag, and the method further comprises:

bonding the constructs to materials in the fluid compartments;

releasing the contents of the fluid compartments into a bulk phase;

capturing the constructs; and

isolating the captured constructs and the materials from other materials in the bulk phase.

28. A barcode library, comprising:

a plurality of fluid compartments, each containing one or more copies of a unique construct, wherein each construct comprises a unique N-mer and a functional N-mer.

29. The library of claim 28, wherein each functional N-mer is one selected from the list consisting of sticky end;

sequence-specific primer;

random N-mer;

antibody;

probe target; and

universal primer site.

30. The library of claim 28, wherein the fluid compartments are water-in-oil droplets.

31. The library of claim 28, wherein the unique N-mer is between 3 and 21 nucleotides in length.