Analysis using microfluidic partitioning devices

Patent metadata

Field	Value
Publication number	US-9364829-B2
Application number	US-94548310-A
Country	US
Kind code	B2
Filing date	Nov 12, 2010
Priority date	Jun 2, 2005
Publication date	Jun 14, 2016
Grant date	Jun 14, 2016

Abstract

The invention relates to methods, reagents and devices for detection and characterization of nucleic acids, cells, and other biological samples. Assay method are provided in which a sample is partitioned into sub-samples, and analysis of the contents of the sub-samples carried out. The invention also provides microfluidic devices for conducting the assay. The invention also provides an analysis method using a universal primers and probes for amplification and detection.

First claim

The invention claimed is:

1. An assay method comprising
(a) using a microfluidic device to distribute a sample into a plurality of sub-samples, wherein each sub-sample is distributed into a reaction site by the action of flow channels, control channels and valves:
(i) said sample comprises a rare cell in a background of other cells, wherein the rare cell is less than 1% of the cells in the sample, and
(ii) at least two sub-samples comprise exactly one cell each, one of which is said rare cell;

(b) providing the same reagents in each sub-sample;
(c) conducting a reaction in each sub-sample; and
(d) analyzing the results of each reaction to detect at least two properties of a cell, at least one of which identifies the rare cell, wherein at least one cell analyzed comprises said rare cell.

2. The method of claim 1 wherein the sample is partitioned into 100-100,000 sub-samples.

3. The method of claim 1 , wherein said analyzing comprises detecting a nucleic acid molecule.

4. The method of claim 3 , wherein said nucleic acid molecule comprises RNA.

5. The method of claim 3 , wherein said analyzing comprises detecting said nucleic acid molecule by amplification.

6. The method of claim 5 , wherein said amplification is by PCR or RT-PCR.

7. The method of claim 1 , wherein at least 99% of the sub-samples contain zero or one cell.

8. The method of claim 1 , wherein the cells are bacterial cells.

9. The method of claim 1 wherein at least one property is the presence or absence in the cell of a nucleic acid having a specified sequence.

10. The method of claim 1 wherein at least one property is other than the presence or absence in the cell of a nucleic acid having a specified sequence.

11. The method of claim 3 , wherein said analyzing comprises determining the nucleic acid sequence of the nucleic acid molecule.

12. The method of claim 3 , wherein said analyzing comprises single nucleotide polymorphism analysis.

13. The method of claim 3 , wherein said analyzing comprises quantification of the nucleic acid molecule.

14. The method of claim 1 , wherein the analyzing comprises detecting the presence of absence of a protein or epitope in the cell or on the cell surface.

15. The method of claim 1 , wherein said analyzing comprises detecting secretion of a protein or non-protein molecule by the cell.

16. The method of claim 1 , wherein said analyzing comprises detecting a metabolic reaction or change in cell metabolism.

17. The method of claim 1 , wherein at least one cell analyzed comprises a fetal cell.

18. The method of claim 1 , wherein at least one cell analyzed comprises a cancer cell.

19. The method of claim 18 , wherein the cancer cell is a disseminated cancer cell obtained from blood.

20. The method of claim 1 , wherein the sample comprises peripheral blood mononuclear cells (PBMC).

21. The method of claim 1 , wherein the rare cell is less than 0.1% of the cells in the sample.

22. The method of claim 1 , wherein the rare cell is less than 0.01% of the cells in the sample.

Assignees

• Heid Christian A
• Daridon Antoine
• Fluidigm Corp

Inventors

• Heid Christian A
• Daridon Antoine

Classifications

• C12Q2525/155

  incorporating/generating a new priming site · CPC title

• B01L7/52

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• B01L3/5027 Primary

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title