Methods for quantitating DNA using digital multiple displacement amplification

• US 10,487,354 B2
• Filed: 09/11/2015
• Issued: 11/26/2019
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: The Broad Institute, Inc.
• Inventors: Blainey, Paul, Xu, Liyi
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US14/851,945
• Publication Number:
  US 20160068899A1
• Time in Patent Office: 1,537 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention relates to methods of quantifying nucleic acids involving (a) contacting a sample to be tested with whole-genome amplification (WGA) reaction components and a dye molecule to form a reaction sample, wherein the dye molecule detects the nucleic acid; (b) partitioning the reaction sample wherein each partitioned reaction sample corresponds to a single reaction; (c) allowing the reaction to occur in the partitioned reaction sample and (d) determining the number of partitioned reaction samples having the dye molecule, wherein the dye molecule indicates the presence of the nucleic acid; thereby quantifying the nucleic acid in the sample to be tested.

Claims

1. A method of quantifying a nucleic acid molecule comprising

(a) contacting a sample to be tested with whole-genome amplification (WGA) reaction components and a label that detects nucleic acid molecules to form a reaction sample;

(b) partitioning the reaction sample at limiting dilution into monodisperse droplets, wherein each droplet corresponds to a single WGA reaction comprising a single nucleic acid molecule or no nucleic acid molecule;

(c) allowing the WGA reaction to occur in the droplets; and

(d) determining the number of droplets comprising nucleic acid molecules by detecting the label, thereby quantifying the nucleic acid in the sample to be tested.

2. The method of claim 1, wherein the nucleic acid is selected from the group consisting of double stranded DNA, single stranded DNA, RNA, and PNA.

3. The method of claim 1, wherein the WGA is carried out by multiple displacement amplification (MDA) and optionally

wherein the WGA reaction components are from a commercial MDA kit selected from the group consisting of Repli-G Midi (Oiagen), GenomiPhi (GE) and RepliPHI (Epicentre).

4. The method of claim 1, wherein the WGA reaction components further comprise an emulsion component and optionally wherein the emulsion component is an inert oil and optionally wherein the oil is a fluorocarbon oil or perfluoropolyether.

5. The method of claim 1, wherein the WGA reaction is supplemented with a surfactant and optionally wherein the surfactant is tween, triton, span, pluronic, a poly(ethylene glycol)-containing molecule or a perfluropolyether-containing molecule and optionally wherein the tween is 0.3% Tween-20.

6. The method of claim 1, wherein the reaction occurs in the partitioned reaction sample at 30°

C. or wherein the reaction occurs in the partitioned reaction sample at 65°

C.

7. The method of claim 1, further comprising a heat lysis reaction and optionally

wherein the heat lysis reaction is at 95°

C.

8. The method of claim 1, wherein the dye molecule is an intercalating dye.

9. The method of claim 1, wherein the dye is a fluorescent dye.

10. The method of claim 9, wherein the dye is an Evagreen dye, a ROX dye, or a SYTOX Orange dye.

11. The method of claim 1, wherein the dye is attached to a nucleic acid hydrolysis probe comprising an energy transfer moiety, wherein the energy transfer moiety is a second dye or quencher, and optionally

wherein the hydrolysis probe is a Taqman probe.

12. The method of claim 1, wherein the method further comprises recovering material from the droplet for further analysis and optionally

wherein the further analysis is PCR or sequencing.

13. The method of claim 1, wherein the sample to be tested comprises bacteria.

14. The method of claim 1, wherein the method reduces WGA amplification bias, reduces incidence of chimeric sequences, or is used as an analytical method to determine the type of template molecules or contaminant molecules in a sample.

15. A method of amplifying a nucleic acid molecule in a sample comprising

(a) contacting a sample with whole-genome amplification (WGA) reaction components and a label that detects nucleic acid molecules to form a reaction sample;

(b) partitioning the reaction sample at limiting dilution into a hydrogel; and

(c) allowing the WGA reaction to occur in the hydrogel.

16. The method of claim 15, wherein the method further comprises recovering a nucleic acid cluster from the hydrogel for further analysis.

17. The method of claim 15, wherein the method reduces WGA amplification bias, reduces incidence of chimeric sequences, or is used as an analytical method to determine type of template molecules or contaminant molecules in a sample.

18. The method of claim 15, further comprising detecting or characterizing labelled nucleic acid clusters within the hydrogel, thereby quantifying the nucleic acid molecules in the sample to be tested, wherein the characterizing is determining the cluster radius, the cluster size, or the optical intensity of the labelled nucleic acid cluster.

19. The method of claim 15, wherein the hydrogel comprises a peptide, a protein, a polysaccharide, a synthetic polymer, a natural polymer and/or a block copolymer.

20. A method of preparing nucleic acid molecules for analysis, the method comprising:

(a) contacting a sample to be tested with whole-genome amplification (WGA) reaction components and a label that detects nucleic acid molecules to form a reaction sample;

(b) partitioning the reaction sample into monodisperse droplets, wherein each droplet corresponds to a single WGA reaction comprising a single nucleic acid molecule or no nucleic acid molecule; and

(c) allowing the WGA reaction to occur in the droplets.