Single-cell nucleic acid analysis

Patent metadata

Field	Value
Publication number	US-9909179-B2
Application number	US-201514977414-A
Country	US
Kind code	B2
Filing date	Dec 21, 2015
Priority date	Jan 13, 2009
Publication date	Mar 6, 2018
Grant date	Mar 6, 2018

Abstract

The present invention provides methods for analysis of genomic DNA and/or RNA from small samples or even single cells. Methods for analyzing genomic DNA can entail whole genome amplification (WGA), followed by preamplification and amplification of selected target nucleic acids. Methods for analyzing RNA can entail reverse transcription of the desired RNA, followed by preamplification and amplification of selected target nucleic acids.

First claim

What is claimed is:

1. A method for analyzing nucleic acids from a single cell, said method comprising:
(a) preamplifying DNA obtained from a single cell using a plurality of target-specific primers to produce a preamplification reaction mixture comprising a plurality of amplicons specific for a plurality of target nucleic acids;
(b) distributing the plurality of amplicons within a device comprising separate chambers;
(c) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers.

2. The method of claim 1 , wherein the DNA comprises genomic DNA, and the method further comprises whole genome amplification prior to step (a).

3. The method of claim 1 , wherein the DNA comprises cDNA.

4. The method of claim 3 , wherein the cDNA is produced by reverse transcription of RNA.

5. The method of claim 4 , wherein the RNA is mRNA.

6. The method of claim 1 , wherein the DNA comprises DNA produced synthetically or by amplification.

7. The method of claim 1 , wherein said single cell comprises a mammalian cell.

8. The method of claim 7 , wherein said single cell is selected from a cell from a preimplantation embryo, a stem cell, a suspected cancer cell, a cell from a pathogenic organism, and a cell obtained from a crime scene.

9. The method of claim 8 , wherein the cell is a suspected cancer cell.

10. The method of claim 1 , wherein no probe is present in the preamplification mixture.

11. The method of claim 1 , wherein said plurality of target-specific primer pairs used for preamplification amplifies one or more single nucleotide polymorphisms (SNPs).

12. The method of claim 1 , wherein the device comprises a microfluidic device fabricated, at least in part, from an elastomeric material.

13. The method of claim 1 , wherein the preamplification and the amplification are carried out by polymerase chain reaction (PCR).

14. The method of claim 1 , wherein the presence of an amplification product is determined by quantitative real-time polymerase chain reaction (qPCR).

15. The method of claim 14 , wherein a universal qPCR probe is employed to detect amplification products.

16. The method of claim 15 , wherein the universal qPCR probe comprises a double-stranded DNA (dsDNA) dye.

17. The method of claim 13 , wherein one or more target-specific qPCR probes is employed to detect amplification products.

18. The method of claim 13 , wherein the presence of an amplification product is detected using a fluorogenic nuclease assay.

19. The method of claim 18 , wherein the presence of an amplification product is detected using a dual-labeled fluorogenic oligonucleotide probe.

20. The method of claim 1 , wherein the plurality of amplicons comprises at least 10 amplicons.

Assignees

• Fluidigm Corp

Inventors

• Hamilton Amy
• Lin Min
• Mir Alain
• Pieprzyk Martin

Classifications

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title

• C12Q2565/101

  Interaction between at least two labels · CPC title

• C12Q2531/137

  Ligase Chain Reaction [LCR] · CPC title

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title

• C12Q2531/113

  PCR · CPC title