METHODS FOR QUANTITATING DNA USING DIGITAL MULTIPLE DISPLACMENT AMPLIFICATION

• US 20160068899A1
• Filed: 09/11/2015
• Published: 03/10/2016
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Blainey, Paul, Xu, Liyi
• Granted Patent: US 10,487,354 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 provides improved methods for detecting contamination in WGA reagents as well as improved methods for quantitating nucleic acids, such as DNA. The present invention relates to novel methods of quantifying nucleic acids involving whole-genome amplification (WGA) reaction components and a dye molecule to detects nucleic acids and partitioning reactions to quantify nucleic acids.

Claims

1. A method of quantifying a nucleic acid comprising (a) contacting a sample to be tested with whole-genome amplification (WGA) reaction components and a dye molecule or other probe 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 is quantitated 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, single stranded RNA or 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, and optionally

wherein the commercial MDA kit selected from the group consisting of Repli-G Midi (Qiagen), 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 partitioned reaction sample is a droplet and optionally

wherein the droplet is a monodisperse aqueous droplet.

6. The method of claim 1,

wherein the partitioned reaction sample is in a matrix and optionally

wherein the matrix is a gel and optionally

wherein the gel is a hydrogel and optionally

wherein the hydrogel comprises a polyethylene glycol, a peptide, a protein, a polysaccharide, a synthetic polymer, a natural polymer and/or a block copolymer.

7. 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.

8. 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.

9. The method of claim 1, further comprising a heat lysis reaction and optionally wherein the heat lysis reaction is at 95°

C.

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

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

13. The method of claim 1,

wherein the dye is attached to a nucleic acid hydrolysis probe comprising an energy transfer moiety such as a second dye or quencher and optionally

wherein the energy transfer moiety is a second dye or a quencher and optionally

wherein the quencher is a Tallman probe.

14. The method of claim 1,

wherein the method further comprises recovering material from the droplet or matrix for further analysis and optionally

wherein the further analysis is PCR or sequencing.

15. The method of claim 1,

wherein the sample to be tested is a bacteria and optionally

wherein the bacteria is a Staphylococcus Aureus or an Escherichia coll.

16. The method of claim 1

wherein the method reduces WGA amplification bias or

wherein the method reduces incidence of chimeric sequences or

wherein the method is used as an analytical method to determine the number and or type of template molecules or contaminant molecules in a sample or

wherein the method is a preparative method to amplify a template or contaminant molecules in a sample prior to further analysis.

12. The method of claim 12,

wherein the dye is an Evagreen dye or

wherein the dye is a ROX dye or

wherein the dye is a SYTOX Orange dye.