COMPOSITIONS AND METHODS FOR MOLECULAR LABELING

• US 20190316119A1
• Filed: 05/01/2019
• Published: 10/17/2019
• 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 L., Olson, Jeffrey Charles, Watson, Andrew, Brown, Keith, Link, Darren R.
• Granted Patent: US 11,168,353 B2
• Time in Patent Office: Days
• 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: 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

1-31. -31. (canceled)

32. A method for analyzing a nucleic acid, the method comprising:

obtaining nucleic acid from a cell or a population of cells;

exposing the nucleic acid to a transposase to form a reaction product;

introducing the reaction product to a droplet comprising barcoded primers and reagents for amplification;

conducting an amplification reaction in the droplet to produce barcoded amplicons corresponding to transposase-accessible regions of the nucleic acid; and

sequencing the transposase-accessible regions.

33. The method of claim 32, wherein the nucleic acid comprises genomic DNA.

34. The method of claim 32, wherein the exposing step results in reaction products in an aqueous suspension.

35. The method of claim 32, wherein the reaction product is a transposed nucleic acid.

36. The method of claim 32, wherein the introducing step comprises forming a plurality of droplets, each comprising a different reaction product.

37. The method of claim 32, wherein the droplet comprises a single molecule of the reaction product.

38. The method of claim 32, wherein the droplet is formed by intersecting an aqueous fluid comprising the reaction product with a counter-propagating stream of oil.

39. The method of claim 32, wherein each of the barcoded primers comprises a functional N-mer portion and a unique N-mer portion.

40. The method of claim 39, wherein all unique N-mers within a droplet are identical.

41. The method of claim 32, wherein the barcoded primers hybridize at random locations on the reaction product.

42. The method of claim 32, wherein the barcoded primers are attached to beads.

43. The method of claim 32, further comprising breaking the droplets after the conducting step.

44. The method of claim 43, wherein breaking the droplets comprises releasing the barcoded amplicons into an aqueous phase.

45. The method of claim 32, wherein the barcoded amplicons are not attached to beads.