Methods and systems for detecting biological components

Patent metadata

Field	Value
Publication number	US-10161007-B2
Application number	US-201314420646-A
Country	US
Kind code	B2
Filing date	Aug 12, 2013
Priority date	Aug 13, 2012
Publication date	Dec 25, 2018
Grant date	Dec 25, 2018

Abstract

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

First claim

What is claimed is:

1. A method of amplifying a target polynucleotide, the method comprising:
(a) contacting on a microfluidic device a biological sample comprising a cell containing a target polynucleotide with lysis reagents in a microdroplet, the lysis reagents comprising an enzyme having protease activity, wherein the microdroplet is encapsulated in an immiscible carrier fluid;
(b) incubating the microdroplet containing the sample and the lysis reagents comprising the enzyme having protease activity for a period of time sufficient to allow the cell to lyse and form a cell lysate and the protease to digest inhibitory proteins;
(c) incubating the microdroplet containing the cell lysate and the enzyme having the protease activity at a temperature sufficient to inactivate the enzyme having protease activity;
(d) adding to the microdroplet containing the cell lysate, amplification reagents to form a microdroplet containing the cell lysate and the amplification reagents in the immiscible carrier fluid; and
(e) amplifying the target polynucleotide within the microdroplet containing the cell lysate and the amplification reagents; wherein the method does not comprise a step of selectively removing reagents from the microdroplet containing the cell lysate and amplification reagents prior to step (e).

2. The method of claim 1 , wherein the enzyme is a protease.

3. The method of claim 1 , wherein the enzyme is proteinase K.

4. The method of claim 1 , wherein the microdroplet containing the cell lysate and the amplification reagents has a volume of 0.001 to 1000 picoliters.

5. The method of claim 1 , wherein the microdroplet containing the cell lysate and the amplification reagents has a diameter of between 0.1 microns and 1000 microns.

6. The method of claim 1 , wherein the microdroplet containing the sample contains a single cell.

7. The method of claim 1 , wherein the step (d) comprises (i) contacting the microdroplet containing the cell lysate with a continuous stream of fluid comprising the amplification reagents, and (ii) forming the microdroplet containing the cell lysate and the amplification reagents from a portion of the continuous stream of fluid and the microdroplet containing the cell lysate.

8. The method of claim 1 , wherein the amplification comprises reverse transcription to produce a reverse transcription product.

9. The method of claim 1 , wherein the amplification comprises polymerase chain reaction.

10. The method of claim 1 , further comprising detecting an amplification product of the target polynucleotide.

11. The method of claim 10 , wherein detecting the amplification product of the target polynucleotide comprises determining a sequence of the amplification product.

12. The method of claim 10 , wherein detecting the amplification product of the target polynucleotide comprises forming a double-emulsion comprising the microdroplet containing amplification product and sorting the double-emulsion based on at least one of droplet size and fluorescence.

13. The method of claim 10 , further comprising the step of sorting the microdroplet based on results of detecting the amplification product of the target polynucleotide.

14. The method of claim 10 , further comprising determining a number of microdroplets containing the target polynucleotide based on results of detecting the amplification product of the target polynucleotide.

15. The method of claim 10 , further comprising determining percentage of microdroplets containing the target polynucleotide based on results of detecting the amplification product of the target polynucleotide.

16. The method of claim 10 , wherein amplifying the target polynucleotide comprises extension of a primer comprising a capture sequence and detecting the target polynucleotide comprises detecting the capture sequence in the amplification product.

17. The method of claim 16 , wherein detecting presence of the capture sequence comprises introducing a fluorescent bead into the microdroplet containing the amplification product, wherein the fluorescent bead comprises a nucleotide sequence complementary to the capture sequence.

18. The method of claim 1 , wherein at least one of the steps (d) and (e) are performed under microfluidic control.

19. The method of claim 1 , wherein step (d) is performed under microfluidic control.

20. The method of claim 1 , wherein the step (d) comprises droplet coalescence.

21. The method of claim 1 , wherein the step (d) comprises picoinjection.

22. The method of claim 1 , further comprising sorting prior to lysing the sample containing target polynucleotide.

23. The method of claim 1 , further comprising sorting between step (a) and step (d).

Assignees

• Univ California

Inventors

• Abate Adam R
• Eastbum Dennis Jay
• Sciambi Adam R

Classifications

• B01L2300/0864

  comprising only one inlet and multiple receiving wells, e.g. for separation, splitting · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title