Compositions and methods for molecular labeling

Patent metadata

Field	Value
Publication number	US-11168353-B2
Application number	US-201916400858-A
Country	US
Kind code	B2
Filing date	May 1, 2019
Priority date	Feb 18, 2011
Publication date	Nov 9, 2021
Grant date	Nov 9, 2021

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 analyzing a nucleic acid, the method comprising:
isolating a cell within a droplet of a plurality of droplets;
lysing the cell within the droplet to release nucleic acid;
exposing the nucleic acid to transposase enzymes and barcoded oligonucleotides in the droplet, wherein the barcoded oligonucleotides comprise a universal priming site and a barcode specific to the droplet;
conducting a transposition reaction with the transposase enzymes in the droplet to produce barcoded fragments of the nucleic acid;
breaking the plurality of droplets; and
amplifying the barcoded fragments to yield barcoded amplicons, wherein the amplifying step incorporates sequencing adaptors into the barcoded amplicons.

2. The method of claim 1 , wherein the nucleic acid comprises genomic DNA.

3. The method of claim 1 , wherein the lysing step includes exposing the cell to heat and/or detergent in the droplet.

4. The method of claim 3 , further comprising sequencing the amplified fragments.

5. The method of claim 3 , wherein each of the plurality of droplets comprises a different set of barcoded fragments.

6. The method of claim 1 , wherein the amplifying step comprises PCR.

7. The method of claim 6 , wherein breaking the droplets comprises releasing the barcoded fragments into an aqueous phase and pooling barcoded products from the plurality of droplets.

8. The method of claim 1 , wherein the barcoded fragments are not attached to beads.

9. The method of claim 7 , further comprising sequencing the barcoded amplicons.

10. A method for analyzing a nucleic acid, the method comprising:
isolating a single cell within one of a plurality of aqueous partitions;
lysing the single cell to release nucleic acid from the single cell;
exposing the nucleic acid to transposase enzymes and oligonucleotides in the one aqueous partition, wherein the oligonucleotides include a primer-binding site and a barcode specific to the one aqueous partition;
conducting a transposition reaction with the transposase enzymes in the partition to produce barcoded segments of the nucleic acid;
releasing contents of partitions; and
amplifying the barcoded segments using primers that include sequencing adaptors and that anneal to the primer-binding site to form barcoded amplicons that include the sequencing adaptors.

11. The method of claim 10 , further comprising sequencing the barcoded amplicons.

Assignees

• Bio Rad Laboratories

Inventors

• Samuels Michael L
• 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 Primary

  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