SYSTEMS AND METHODS FOR BARCODING NUCLEIC ACIDS

• US 20150298091A1
• Filed: 06/09/2015
• Published: 10/22/2015
• Est. Priority Date: 04/21/2014
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Vilnius University
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Vilnius University
• Inventors: Weitz, David A., Kirschner, Marc W., Klein, Allon Moshe, Akartuna, Ilke, Mazutis, Linas
• Application Number:
  US14/734,903
• Publication Number:
  US 20150298091A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/0858 Side walls
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present invention generally relates to microfluidics and labeled nucleic acids. For example, certain aspects are generally directed to systems and methods for labeling nucleic acids within microfluidic droplets. In one set of embodiments, the nucleic acids may include “barcodes” or unique sequences that can be used to distinguish nucleic acids in a droplet from those in another droplet, for instance, even after the nucleic acids are pooled together. In some cases, the unique sequences may be incorporated into individual droplets using particles and attached to nucleic acids contained within the droplets (for example, released from lysed cells). In some cases, the barcodes may be used to distinguish tens, hundreds, or even thousands of nucleic acids, e.g., arising from different cells or other sources.

Claims

1. A method of barcoding nucleic acids, the method comprising:

encapsulating a cell and a particle within a microfluidic droplet, the particle having attached thereto a barcoded nucleic acid;

cleaving the barcoded nucleic acid from the particle to release the barcoded nucleic acid into the microfluidic droplet;

lysing the cell within the microfluidic droplet to release cellular nucleic acid from the cell into the microfluidic droplet; and

attaching the released cellular nucleic acid to the released barcoded nucleic acid within the microfluidic droplet.

2. The method of claim 1, wherein the particle is a hydrogel particle.

3. The method of claim 1, comprising photocleaving the barcoded nucleic acid from the particle.

4. The method of claim 1, comprising encapsulating only a single cell and only a single particle within the microfluidic droplet.

5. The method of claim 1, wherein the microfluidic droplet is one of a plurality of microfluidic droplets, wherein, after encapsulation, the microfluidic droplets contain cells and particles such that at least about 90% of the plurality of microfluidic droplets contains only one particle and at least about 90% of the plurality of microfluidic droplets contains only one cell.

6. The method of claim 5, wherein, after cleavage, at least 90% of the microfluidic droplets contains only a single type of barcoded nucleic acid.

7. The method of claim 5, further comprising breaking at least some of the microfluidic droplets to release and combine the attached cellular and barcoded nucleic acids.

8. The method of claim 7, further comprising amplifying and/or sequencing the attached cellular and barcoded nucleic acids.

9. The method of claim 1, wherein after cleavage, the droplet has a concentration of the barcoded nucleic acids of at least 1 micromolar.

10. The method of claim 1, wherein at least some of the barcoded nucleic acids, prior to cleaving, are covalently bonded to the particles via an acrylic phosphoramidite linkage, an amino linkage, or a biotin-steptavidin linkage.

11. The method of claim 1, comprising enzymatically attaching the released cellular nucleic acid to the released barcoded nucleic acid.

12. A method of containing barcoded nucleic acids within microfluidic droplets, the method comprising:

attaching first oligonucleotides to a plurality of particles such that at least about 90% of the particles has covalently bonded thereto only one first oligonucleotide, wherein the first oligonucleotides are taken from a first pool of at least 100 unique oligonucleotides;

attaching second oligonucleotides to the first oligonucleotides such that at least about 90% of the first oligonucleotides has covalently bonded thereto only one second oligonucleotide, wherein the second oligonucleotides are taken from a second pool of at least 100 unique oligonucleotides;

encapsulating the plurality of particles within a plurality of microfluidic droplets such that at least about 90% of the plurality of microfluidic droplets contains only one particle; and

cleaving the first and second oligonucleotides from the particles into the microfluidic droplets.

13. The method of claim 12, wherein the particle is a hydrogel particle.

14. The method of claim 12, wherein at least some of the barcoded nucleic acids, prior to cleaving, are covalently bonded to the particles via an acrylic phosphoramidite linkage, an amino linkage, or a biotin-steptavidin linkage.

15. The method of claim 12, wherein the first pool comprises at least 1,000 unique oligonucleotides and the second pool comprises at least 1,000 unique oligonucleotides.

16. The method of claim 12, further comprising encapsulating a plurality of cells in the plurality of microfluidic droplets such that at least about 90% of the plurality of microfluidic droplets contains only one cell.

17. The method of claim 12, wherein after cleavage, the droplets containing particles have an average concentration of the first and second oligonucleotides of at least 1 micromolar.

18. An article for barcoding nucleic acids, comprising:

a plurality of at least 10,000 microfluidic droplets, at least some of the droplets containing cell lysate comprising nucleic acid fragments, a plurality of the nucleic acid fragments within a droplet being bound to an oligonucleotide tag, wherein the oligonucleotide tag within the droplet is distinguishable from oligonucleotide tags within the other droplets of the plurality of 10,000 microfluidic droplets.

19. The article of claim 18, wherein the microfluidic droplets comprise a plurality of at least distinguishable 1,000,000 oligonucleotide tags within the microfluidic droplets.

20. The article of claim 18, wherein at least some of the distinguishable oligonucleotide tags each comprise a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotides are taken from a first pool of at least 100 unique first oligonucleotides, and wherein the second oligonucleotides are taken from a second pool of at least 100 unique first oligonucleotides.

21. The article of claim 18, wherein in at least about 90% of the plurality of microfluidic droplets, the cell lysate arises from only one cell.