Methods and systems for detecting biological components

Patent metadata

Field	Value
Publication number	US-2016177375-A1
Application number	US-201615047555-A
Country	US
Kind code	A1
Filing date	Feb 18, 2016
Priority date	Aug 13, 2012
Publication date	Jun 23, 2016
Grant date	—

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 one or more target polynucleotides, the method comprising:
(a) lysing one or more polynucleotide-containing components from a sample in a droplet to form a lysate in a lysate droplet, wherein the lysate comprises the one or more target polynucleotides and the lysate droplet is in an immiscible carrier fluid; (b) adding to the lysate reagents for performing a nucleic acid amplification reaction to form an amplification mixture in an amplification droplet, wherein the amplification droplet is in the immiscible carrier fluid; and (c) amplifying the one or more target polynucleotides in the amplification droplet; wherein no reagents are selectively removed from the lysate droplet or from the amplification droplet prior to step (c).

2 . The method of claim 1 , wherein (i) the amplification droplet has a volume of 0.001 to 1000 picoliters, or (ii) the amplification droplet has a diameter of between 0.1 microns to 1000 microns.

3 . The method of claim 1 , wherein the lysate droplet contains lysate of a single cell.

4 . The method of claim 1 , wherein step (b) comprises (i) merging the lysate droplet with a stream of fluid comprising the reagents for performing a nucleic acid amplification reaction, and (ii) forming the amplification droplet from the stream of fluid.

5 . The method of claim 1 , wherein the reagents for performing a nucleic acid amplification reaction are added to the lysate droplet by droplet coalescence or picoinjection.

6 . The method of claim 1 , wherein the one or more target polynucleotides are DNA.

7 . The method of claim 1 , wherein the one or more target polynucleotides are RNA.

8 . The method of claim 7 , wherein amplification comprises reverse transcription to produce a reverse transcription product.

9 . The method of claim 1 , further comprising the step of detecting presence of an amplification product amplified in step (c).

10 . The method of claim 9 , wherein detecting presence of an amplification product comprises sequencing the amplification product.

11 . The method of claim 9 , wherein detecting presence of an amplification product comprises forming a double-emulsion comprising an amplification droplet within an outer droplet, and sorting the double-emulsion based on droplet size and fluorescence.

12 . The method of claim 9 , further comprising the step of sorting the amplification droplet based on results of the detection step.

13 . The method of claim 9 , further comprising determining the number or percentage of cells containing the one or more target polynucleotides based on results of the detection step.

14 . The method of claim 9 , wherein amplification comprises extension of a primer comprising a capture sequence, and detecting presence of an amplification product comprises detecting the capture sequence in the amplification product.

15 . The method of claim 1 , wherein one or more steps are performed under microfluidic control.

16 . The method of claim 1 , wherein step (b) comprises applying an electrical field to the lysate droplet.

17 . The method of claim 1 , wherein lysing the one or more polynucleotide-containing components comprises exposure to a protease, and further wherein the protease is inactivated prior to step (c).

18 . A method of amplifying one or more target polynucleotides, the method comprising:
(a) lysing one or more polynucleotide-containing components from a sample with a protease in a droplet to form a lysate in a lysate droplet, wherein the lysate comprises the one or more target polynucleotides and the lysate droplet is in an immiscible carrier fluid; (b) adding to the lysate reagents for performing a nucleic acid amplification reaction to form an amplification mixture in an amplification droplet, wherein the amplification droplet is in the immiscible carrier fluid; and (c) amplifying the one or more target polynucleotides in the amplification droplet; wherein the protease of step (a) is inactivated during or prior to step (c).

19 . The method of claim 18 , wherein (i) the amplification droplet has a volume of 0.001 to 1000 picoliters, or (ii) the amplification droplet has a diameter of between 0.1 microns to 1000 microns.

20 . The method of claim 18 , wherein the lysate droplet contains lysate of a single cell.

Assignees

• Univ California

Inventors

• Abate Adam R
• Eastburn Dennis Jay
• Sciambi Adam R

Classifications

• B01L7/52

  with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• B01L2300/0864

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

• C12Q2563/159

  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 title