Single cell nucleic acid analysis

• US 8,628,923 B2
• Filed: 01/13/2010
• Issued: 01/14/2014
• Est. Priority Date: 01/13/2009
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Hamilton, Amy, Lin, Min, Mir, Alain, Pieprzyk, Martin
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/687,018
• Publication Number:
  US 20100178655A1
• Time in Patent Office: 1,462 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

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.

Claims

1. A method for analyzing DNA from a single cell, said method comprising:

(a) performing whole genome amplification of the genome of a single cell to produce an amplified genome, wherein the method entails performing whole genome amplification for fewer than, or about, 10 polymerase chain reaction (PCR) cycles;

(b) preamplifying the amplified genome using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;

(c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification; and

(d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers.

6. The method of any of claim 1, 2, or 3-5, wherein said single cell comprises a mammalian cell.

7. The method of claim 6, 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.

8. The method of claim 7, wherein the cell comprises a human blastomere.

9. The method of claim 8, wherein the human blastomere is from an eight-cell stage embryo.

10. The method of claim 7, wherein the cell comprises a human stem cell.

12. The method of claim 1 or 4, wherein said whole genome amplification is performed for more than two amplification cycles.

14. The method of claim 12, wherein said whole genome amplification is performed for between four and eight cycles, inclusive.

15. The method of claim 1 or 4, wherein said whole genome amplification is carried out using a technique selected from the group consisting of primer extension PCR (PEP), degenerated oligonucleotide primed PCR, and ligation-mediated PCR (LMP).

20. The method of claim 1 or 5, wherein said preamplification is carried out for 8-18 cycles.

21. The method of any of claim 1, 2, or 3-5, wherein said amplification is carried out using one or more primer pairs specific for said one or more target nucleic acids.

24. The method of claim 1, 2, or 3-5, wherein said plurality of target-specific primer pairs used for preamplification amplifies one or more single nucleotide polymorphisms (SNPs).

25. The method of claim 24, wherein the presence of said one or more SNPs is/are correlated with the presence of one or more genetic defects.

26. The method of any of claim 1, 2, or 3-5, wherein the microfluidic device is fabricated, at least in part, from an elastomeric material.

27. The method of any of claim 1, 2, or 3-5, wherein the preamplification and the amplification is carried out by polymerase chain reaction (PCR).

28. The method of any of claim 1, 2, or 3-5, wherein the presence of an amplification product is determined by quantitative real-time polymerase chain reaction (qPCR).

29. The method of claim 28, wherein a universal qPCR probe is employed in the amplification mixtures to detect amplification products.

30. The method of claim 29, wherein the universal qPCR probe comprises a double-stranded DNA (dsDNA) dye.

31. The method of claim 28, wherein one or more target-specific qPCR probes is employed in the amplification mixtures to detect amplification products.

32. The method of claim 28, wherein the presence of an amplification product is detected using a fluorogenic nuclease assay.

33. The assay method of claim 32, wherein the presence of an amplification product is detected using a dual-labeled fluorogenic oligonucleotide probe.

34. The method of any of claim 1, 2, or 3-5, wherein the plurality of amplicons comprises at least 10 amplicons.

35. The method of any of claim 1, 2, or 3-5, wherein the plurality of amplicons comprises from 10 to 96 amplicons.

2. A method for analyzing mRNA from a single cell, said method comprising:

(a) preparing DNA from the mRNA of a single cell;

(b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids, wherein said preamplification is carried out for 18 polymerase chain reaction (PCR) cycles or fewer;

(c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification; and

(d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein the amplicons are detected by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.

16. The method of claim 2, wherein the DNA is cDNA produced by reverse transcription of mRNA.

22. The method of claim 16, wherein DNA is produced by reverse transcription, which is followed by preamplification in the same reaction mixture.

23. The method of claim 16, wherein no probe is present in the preamplification mixture.

3. A method for analyzing non-coding RNA from a single cell, said method comprising:

(a) preparing DNA from the non-coding RNA of a single cell;

(b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;

(c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification; and

(d) amplifying and detecting the plurality of amplicons wherein different amplicons are amplified and detected in separate chambers, and wherein the amplicons are detected by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.

4. A method for analyzing DNA from a single cell, said method comprising:

(a) performing whole genome amplification of the genome of a single cell to produce an amplified genome, wherein the whole genome amplification comprises polymerase chain reaction (PCR) cycles;

(b) preamplifying the amplified genome using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;

(c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification; and

(d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein primer pairs used in preamplification and/or amplification can amplify and detect single nucleotide polymorphisms (SNPs), by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.

11. The method of claim 4, wherein said whole genome amplification is carried out such that a reaction plateau is not reached.

13. The method of claim 4, wherein said whole genome amplification is performed for fewer than, or about, 10 amplification cycles.

5. A method for analyzing RNA from a single cell, said method comprising:

(a) preparing DNA from the RNA of a single cell;

(b) preamplifying the DNA using a plurality of target-specific primer pairs to produce a preamplification reaction mixture comprising a plurality of amplicons specific for one or more target nucleic acids;

(c) distributing the preamplification reaction mixture into separate chambers of a microfluidic device prior to amplification; and

(d) amplifying and detecting the plurality of amplicons, wherein different amplicons are amplified and detected in separate chambers, and wherein primer pairs used in preamplification and/or amplification can amplify and detect single nucleotide polymorphisms (SNPs), by a method selected from a real-time quantitation method, monitoring the amount of amplification product after a predetermined number of cycles, and melting curve analysis.

17. The method of claim 5, wherein the RNA comprises non-coding RNA.

18. The method of claim 17 or 3, wherein the non-coding RNA is selected from the group consisting of small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), and Piwi-interacting RNAs (piRNA).

19. The method of claim 17 or 3, wherein DNA is produced from the non-coding RNA by reverse transcription or amplification.