Compositions and methods for molecular labeling

Patent metadata

Field	Value
Publication number	US-11965877-B2
Application number	US-201514874553-A
Country	US
Kind code	B2
Filing date	Oct 5, 2015
Priority date	Feb 18, 2011
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

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.

First claim

What is claimed is:

1. A method for barcoding RNA from a single cell, the method comprising:
forming a plurality of aqueous droplets in oil, each droplet comprising a plurality of different RNA molecules from a single cell and a plurality of nucleic acid constructs not attached to microbeads, wherein each of the nucleic acid constructs comprises a universal priming site, a barcode sequence and a poly(dT) sequence, wherein the barcode sequence is a unique barcode that allows sequence reads to be assigned to one molecule from a sample;
hybridizing at least one nucleic acid construct to at least one RNA molecule in at least one of the droplets to form at least one hybridized nucleic acid construct that is not attached to a microbead;
extending the at least one hybridized nucleic acid construct that is not attached to the microbead to form an extension product that is not attached to a microbead; and
amplifying, using the universal priming site, the extension product to yield an amplified product.

2. The method of claim 1 , wherein the method includes cell lysis within the droplets using a temperature-inducible protease.

3. The method according to claim 1 , further comprising releasing the extension product from the droplet.

4. The method according to claim 3 , wherein the method further comprises sequencing the amplified product.

5. The method according to claim 1 , wherein the forming step further comprises merging aqueous droplets each comprising a plurality of the RNA molecules with aqueous droplets each comprising a plurality of the nucleic acid constructs.

6. A method for barcoding a target sequence from a single cell, the method comprising:
introducing a plurality of nucleic acid constructs not attached to microbeads into a plurality of aqueous droplets in oil, each droplet containing a plurality of RNA molecules from a single cell and a plurality of the nucleic acid constructs not attached to microbeads, wherein each of the nucleic acid constructs within the same droplet comprises a universal priming site, a barcode sequence, and a random hexamer, wherein the barcode sequence is a unique barcode that allows sequence reads to be assigned to one molecule from the cell;
hybridizing a nucleic acid construct to a target sequence in at least one droplet to form a hybridized nucleic acid construct that is not attached to a microbead;
extending the hybridized nucleic acid construct to produce a barcoded extension product not attached to a microbead; and
amplifying, using the universal priming site, the barcoded extension product to yield an amplified product.

7. The method of claim 6 , wherein the method includes cell lysis within the droplets using a temperature-inducible protease.

8. The method of claim 6 , wherein the extension product comprises a barcoded cDNA sequence.

9. The method of claim 6 , further comprising releasing the at least one hybridized nucleic acid construct from the at least one aqueous droplet in oil before the extending step.

10. A method for barcoding RNA from a single cell, the method comprising:
forming a plurality of aqueous droplets in oil, each droplet comprising a plurality of different RNA molecules from a single cell and a plurality of nucleic acid constructs, wherein each of the nucleic acid constructs comprises a universal priming site, a sequence specific region, and a unique barcode that allows sequence reads to be assigned to one molecule from a sample;
hybridizing at least one nucleic acid construct to at least one RNA molecule in at least one of the droplets;
extending the hybridized nucleic acid construct to produce an extension product not attached to a microbead; and
amplifying, using the universal priming site, the extension product to yield an amplified product.

11. The method of claim 10 , wherein the method includes cell lysis within the droplets using a temperature-inducible protease.

12. The method according to claim 10 , wherein the method further comprises releasing the extension product from the droplets prior to the amplifying step.

13. The method according to claim 12 , further comprising sequencing the amplified product.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Samuels Michael
• Olson Jeffrey Charles
• Watson Andrew
• Brown Keith
• Link Darren R

Classifications

• C12N15/1075 Primary

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title

• C12Q2521/10

  Nucleotidyl transfering · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• G01N33/53 Primary

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title