System and method for determining copies-per-unit-volume using pcr and flow control of droplets

Patent metadata

Field	Value
Publication number	US-2016208342-A1
Application number	US-201615080801-A
Country	US
Kind code	A1
Filing date	Mar 25, 2016
Priority date	Apr 1, 2011
Publication date	Jul 21, 2016
Grant date	—

Abstract

Methods and systems for quantification of a target nucleic acid in a sample are provided. The method includes forming a plurality of discrete sample portions. Each of the plurality of discrete sample portions comprising a portion of the sample, and a reaction mixture. The method further includes amplifying the plurality of discrete sample portions to form a plurality of discrete processed sample portions. At least one discrete processed sample portion containing nucleic acid amplification reaction products. Fluorescence signals are detected from the at least one of the plurality of discrete processed sample portions to determine a presence of the at least one target nucleic acid. The method also includes determining the respective volumes of the plurality of the plurality of discrete processed sample portions, and estimating the number of copies-per-unit-volume of the at least one target nucleic acid in the sample. Estimating the number of copies-per-unit-volume is based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.

First claim

1 . A method for quantification of a target nucleic acid in a sample, the method comprising:
forming a plurality of discrete sample portions, each of the plurality of discrete sample portions comprising a portion of the sample, and a reaction mixture, wherein the plurality of discrete sample portions comprises discrete sample portions of a plurality of sizes; amplifying the plurality of discrete sample portions to form a plurality of discrete processed sample portions including at least one discrete processed sample portion containing nucleic acid amplification reaction products; detecting fluorescence signals from the at least one of the plurality of discrete processed sample portions to determine a presence of the at least one target nucleic acid; determining the respective volumes of the plurality of the plurality of discrete processed sample portions; and estimating the number of copies-per-unit-volume of the at least one target nucleic acid in the sample based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.

2 . The method of claim 1 , wherein the plurality of discrete sample portions comprises discrete sample portions of substantially two different sizes.

3 . The method of claim 1 , wherein the plurality of discrete sample portions comprise discrete sample portions of substantially a plurality of predetermined sizes.

4 . The method of claim 1 , wherein each of the plurality of sample portions is at least partially surrounded by a medium that is at least substantially immiscible with the plurality of discrete sample portions.

5 . The method of claim 4 , wherein the medium that is substantially immiscible with the plurality of discrete sample portions comprises at least one selected from the group consisting of: a mineral oil, a silicone oil, a paraffin oil, a fluorinated fluid, a perfluorinated polyether.

6 . The method of claim 1 , wherein the plurality of discrete sample portions comprises porous beads.

7 . The method of claim 1 , wherein the plurality of discrete sample portions comprises magnetic beads.

8 . The method of claim 7 , further comprising magnetically focusing the magnetic beads within a flow stream in a flow cytometer.

9 . The method of claim 1 , wherein determining the respective volumes comprises imaging the plurality of discrete processed sample portions.

10 . A system for quantification of a target nucleic acid in a sample, the system comprising:
an emulsion apparatus configured to form a plurality of discrete sample portions, each of the plurality of discrete sample portions comprising a portion of the sample, and a reaction mixture; an amplification apparatus configured to amplify the plurality of discrete sample portions to form a plurality of discrete processed sample portions including at least one discrete processed sample portion containing nucleic acid amplification reaction products; an excitation detection apparatus configured to detect fluorescence signals from the at least one of the plurality of discrete processed sample portions to determine a presence of the at least one target nucleic acid, wherein the excitation detection apparatus is further configured to determine the respective volumes of the plurality of the plurality of discrete processed sample portions; and a processor configured to estimate the number of copies-per-unit-volume of the at least one target nucleic acid in the sample based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.

11 . The system of claim 10 , further comprising an optical imager configured to determine the respective volumes of the plurality of the plurality of discrete processed sample portions.

12 . The system of claim 10 , wherein the emulsion apparatus is configured to form the plurality of discrete sample portions of a plurality of sizes.

13 . The system of claim 10 , wherein the emulsion apparatus is configured to form the plurality of discrete sample portions of substantially two different sizes.

14 . The system of claim 10 , wherein the emulsion apparatus is configured to form the plurality of discrete sample portions of substantially a plurality of predetermined sizes.

15 . The system of claim 10 , wherein each of the plurality of discrete sample portions is at least partially surrounded by a medium that is at least substantially immiscible with the plurality of discrete sample portions.

16 . The system of claim 15 , wherein the medium that is substantially immiscible with the plurality of sample portions comprises at least one selected from the group consisting of: a mineral oil, a silicone oil, a paraffin oil, a fluorinated fluid, a perfluorinated polyether.

17 . The system of claim 10 , wherein the plurality of discrete sample portions comprises porous beads.

18 . The system of claim 10 , wherein the plurality of discrete sample portions comprises magnetic beads.

19 . The system of claim 18 , wherein the excitation detection apparatus includes a flow cytometer configured to magnetically focus the magnetic beads within a flow stream.

20 . The system of claim 10 , wherein determining the respective volumes comprises imaging the plurality of discrete processed sample portions.

Assignees

• Life Technologies Corp

Inventors

• Janaway Gordon A
• Andersen Mark
• Zgonc Kornelija
• Pallas Michael C
• Sikora Marcin
• Mcfarland Casey R
• Le Ferrier N
• Wang Haopeng
• Gong Jian
• Padmabandu Gothami

Classifications

• C12Q1/6886 Primary

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

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2600/158

  Expression markers · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title