COMPOSITIONS AND METHODS FOR MOLECULAR LABELING

• US 20160289670A1
• Filed: 10/05/2015
• Published: 10/06/2016
• 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 R.
• Granted Patent: US 11,965,877 B2
• Time in Patent Office: Days
• 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

1-54. -54. (canceled)

55. A method for barcoding RNA from a single cell, the method comprising:

introducing a plurality of nucleic acid constructs into a fluid partition comprising a plurality of RNA molecules from a single cell, wherein each of the nucleic acid constructs comprises a barcode correlated with the cell;

hybridizing at least one nucleic acid construct to at least one RNA molecule; and

producing at least one barcoded RNA complex.

56. The method of claim 55, wherein the barcode is the same for all the nucleic acid constructs in the fluid partition.

57. The method according to claim 55, wherein prior to the introducing step, the method further comprising:

forming a fluid partition comprising the single cell; and

lysing the cell within the fluid partition, thereby releasing a plurality of RNA molecules from the cell.

58. The method according to claim 55, wherein prior to the introducing step, the method comprises:

extracting a plurality of RNA molecules from a single cell; and

forming a fluid partition comprising the plurality RNA molecules.

59. The method according to claim 55, wherein the RNA is unlabeled prior to being introduced into the fluid partition.

60. The method according to claim 55, further comprising releasing the barcoded RNA complex from the fluid partition.

61. The method according to claim 55, wherein the method further comprises extending the nucleic acid construct of the barcoded RNA complex to produce a barcoded cDNA sequence.

62. The method according to claim 61, wherein the method further comprises amplifying the barcoded cDNA sequence.

63. The method according to claim 62, further comprising sequencing the barcoded cDNA sequence.

64. The method according to claim 55, wherein the fluid partition is an aqueous droplet surrounded by an immiscible fluid.

65. The method according to claim 64, wherein introducing comprises merging an aqueous droplet comprising a plurality of RNA molecules with an aqueous droplet comprising a plurality of the nucleic acid constructs.

66. The method according to claim 55, wherein the nucleic acid construct comprises a poly(T) sequence.

67. The method according to claim 55, wherein the nucleic acid construct is attached to a solid support.

68. The method according to claim 55, wherein the RNA is the transcribed RNA of the single cell.

69. The method of claim 55, wherein the nucleic acid construct comprises a unique N-mer potion and a functional N-mer portion

70. The method of claim 69, wherein the functional N-mer is a complementary nucleic acid to the RNA.

71. The method of claim 69, wherein the nucleic acid construct comprises a universal priming site, a unique N-mer, and a sequence specific region.

72. A method for barcoding a target sequence from a single cell, the method comprising:

introducing a plurality of nucleic acid constructs into a plurality of fluid partitions each partition comprising a plurality of target sequences from a single cell, wherein each of the nucleic acid constructs within the same fluid partition comprises a barcode sequence correlated with the cell;

hybridizing at least one nucleic acid construct to the target sequence in a fluid partition; and

producing a barcoded target complex in the fluid partition.

73. The method of claim 72, wherein the nucleic acid constructs in different fluid partitions comprise different barcode sequences.

74. The method of claim 72, wherein the target sequence is an RNA molecule.

75. The method of claim 72, wherein the target sequence is a genomic DNA molecule.

76. The method of claim 75, wherein a plurality of nucleic acid constructs hybridize to different sites of the target sequence.

77. The method of claim 72, wherein the nucleic acid construct is generated from ligation of a barcode sequence.