Compositions and methods for molecular labeling

• US 9,150,852 B2
• Filed: 02/16/2012
• Issued: 10/06/2015
• 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, Olson, Jeffrey Charles, Watson, Andrew, Brown, Keith, Link, Darren Roy
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Weiler, Karen S
• Application Number:
  US13/398,677
• Publication Number:
  US 20120220494A1
• Time in Patent Office: 1,328 Days
• Field of Search: None
• US Class Current: 1/1
• 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 first nucleic acid construct wherein the first construct comprises a forward sequence specific primer, a first barcode sequence, and a first universal primer recognition site;

compartmentalizing the first construct into a fluid compartment;

introducing a second nucleic acid construct into the fluid compartment, wherein the second construct comprises a reverse sequence specific primer, a second barcode sequence, and a second universal primer recognition site;

introducing a third construct into the fluid compartment and hybridizing the third construct to the first construct to form a hybrid construct; and

ligating the hybrid construct to form a new construct.

2. The method of claim 1, wherein the constructs are within the fluid compartment in the absence of any target to make a barcode library.

3. The method of claim 1, wherein the first construct and the second construct each comprises a sticky end.

4. The method of claim 1, wherein the fluid compartment is a water-in-oil droplet.

5. The method of claim 1, further comprising:

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

(b) repeating step (a) M times, thereby producing MxN fluid compartments.

6. The method of claim 1, wherein introducing comprises merging droplets in a microfluidic device.

7. The method of claim 1, wherein either the first or second construct further comprises a sequencing platform-specific adaptor.

8. The method of claim 1, further comprising using either the hybrid or second construct in digital PCR.

9. The method of claim 1, wherein each of the barcode sequences is between 3 and 30 nucleotides in length.

10. The method of claim 1, wherein the hybrid or second construct is linked to an antibody.

11. The method of claim 1, wherein the first and second barcode sequences are the same.

12. The method of claim 1, wherein the first and second barcode sequences are different.

13. The method of claim 1, wherein the first and second universal primer recognition sites are the same.

14. The method of claim 1, wherein the first and second universal primer recognition sites are different.