PCR-activated sorting (PAS)

• US 10,697,007 B2
• Filed: 06/25/2015
• Issued: 06/30/2020
• Est. Priority Date: 06/27/2014
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Abate, Adam R., Eastburn, Dennis Jay, Sciambi, Adam R., Lim, Shaun
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US15/317,393
• Publication Number:
  US 20170121756A1
• Time in Patent Office: 1,832 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2563/107 fluorescence
• 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 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.

Claims

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

obtaining a heterogeneous nucleic acid sample comprising nucleic acids;

encapsulating, in a plurality of microdroplets, the nucleic acids, at least one nucleic acid synthesis reagent and at least one detection component, the plurality of microdroplets comprising an aqueous phase fluid in an immiscible phase carrier fluid;

incubating the plurality of microdroplets under conditions that promote nucleic acid synthesis reagent activity;

positioning the plurality of microdroplets in an aqueous phase carrier fluid to provide a plurality of aqueous phase-in-immiscible phase-in aqueous phase microdroplets; and

sorting the plurality of aqueous phase-in-immiscible phase-in aqueous phase microdroplets based on identifying the at least one detection component, to produce sorted microdroplets; and

analyzing at least one unamplified nucleic acid in at least one sorted microdroplet of the sorted microdroplets.

2. The method of claim 1, further comprising identifying a microbial infection based on results of the analyzing.

3. The method of claim 1, wherein the at least one nucleic acid synthesis reagent amplifies only a subset of the nucleic acids.

4. The method of claim 1, wherein the at least one unamplified nucleic acid is characterized by a length of greater than 100 kb.

5. The method of claim 4, wherein the heterogeneous nucleic acid sample comprises an uncultivated microbe.

6. The method of claim 1, further comprising lysing a cell to release the nucleic acids prior to adding the nucleic acid synthesis reagent and detection component to the plurality of microdroplets.

7. The method of claim 6, wherein lysing the cell comprises exposing the cell to a protease, and wherein the method further comprises inactivating the protease prior to the amplification process.

8. The method of claim 6, wherein encapsulating comprises (i) merging a plurality of sample-containing microdroplets to a stream of fluid comprising the nucleic acid synthesis reagent and detection component, and (ii) recovering the plurality of microdroplets from the stream of fluid.

9. The method of claim 8, wherein the method does not comprise a step of selectively removing reagents from the plurality of sample-containing microdroplets or from the plurality of microdroplets.

10. The method of claim 1, wherein the nucleic acids comprise DNA.

11. The method of claim 1, wherein the nucleic acids comprise RNA.

12. The method of claim 1, wherein the nucleic acid synthesis activity comprises reverse transcription.

13. The method of claim 1, wherein the nucleic acid synthesis activity comprises nucleic acid amplification.

14. The method of claim 1, wherein the sorting comprises sorting the plurality of aqueous phase-in-immiscible phase-in aqueous phase microdroplets via Fluorescence-Activated Cell Sorting (FACS).

15. The method of claim 1, wherein the sorting comprises sorting the plurality of aqueous phase-in-immiscible phase-in aqueous phase microdroplets via dielectrophoresis.

16. The method of claim 1, wherein analyzing comprises analyzing epigenetic marks.

17. The method of claim 1, wherein analyzing comprises sequencing.

18. The method of claim 1, wherein analyzing unamplified nucleic acids comprises analyzing unamplified nucleic acids in microdroplets which do not comprise nucleic acid synthesis products.

19. The method of claim 1, wherein analyzing unamplified nucleic acids comprises analyzing unamplified nucleic acids in microdroplets which do comprise nucleic acid synthesis product.