Multiple displacement amplification

Patent metadata

Field	Value
Publication number	US-9890408-B2
Application number	US-90581910-A
Country	US
Kind code	B2
Filing date	Oct 15, 2010
Priority date	Oct 15, 2009
Publication date	Feb 13, 2018
Grant date	Feb 13, 2018

Abstract

The present invention provides methods kits and systems for performing multiple displacement amplification reactions. In one method a sample of nucleic acid is provided. The nucleic acid is contacted with a reaction mixture which includes a set of oligonucleotide primers, a one or more polymerase enzymes and a detergent. The reaction mixture is then subjected to conditions under which the nucleic acid sequence is amplified to produce an amplified product in a multiple displacement reaction. The method may also be carried out by contacting the nucleic acid with the reaction mixture in the form of an emulsion. A kit is also provided for carrying out either the methods described above. The kit includes one or more polymerases, a plurality of primers and a detergent. The kit may also include a hydrophobic polymer and may include instructions for performing a multiple displacement amplification reaction on a nucleic acid sample.

First claim

What is claimed is:

1. A method comprising:
contacting a sample of nucleic acid with a reaction mixture comprising a set of oligonucleotide primers, one or more polymerase enzymes, and one or more water-in-oil emulsion-forming components wherein said emulsion-forming components comprise polysorbate 40 and polydimethylsiloxane such that said sample and said reaction mixture form an emulsion;
subjecting said emulsion to a multiple displacement amplification reaction comprising thermocycling the emulsion, wherein a nucleic acid sequence in said sample is amplified within at least one emulsion micro-droplet to produce at least one multiple displacement amplification product; and
converting said emulsion to aqueous and hydrophobic phases upon completion of said multiple displacement amplification reaction by adding an emulsion-breaking compound that is chloroform to said reaction.

2. The method of claim 1 wherein said reaction mixture further comprises betaine, trehalose, or both betaine and trehalose.

3. The method of claim 2 wherein said betaine is present in said reaction mixture at a concentration between about 0.2 M to about 1.6 M.

4. The method of claim 2 wherein said trehalose is present in said reaction mixture at a concentration between about 0.1 M and 1.0 M.

5. The method of claim 4 wherein said one or more polymerase enzymes comprise Phi29 polymerase, Bst DNA polymerase, and/or Pol I polymerase.

6. The method of claim 5 wherein said reaction mixture further comprises between about 0.2 units/μL to about 0.6 units/μL of Phi29 polymerase, if said Phi29 polymerase is present in said reaction mixture.

7. The method of claim 5 wherein said reaction mixture further comprises between about 0.005 units/μL to about 0.015 units/μL of Pol I polymerase, if said Pol I polymerase is present in said reaction mixture.

8. The method of claim 1 wherein said one or more polymerase enzymes comprise 5′→3′ DNA polymerase activity, 3′→5′ exonuclease activity, and/or 5′→3′ excision repair activity.

9. The method of claim 1 further comprising amplifying a targeted segment of said amplified product to obtain a second amplification product.

10. The method of claim 9 further comprising determining the base composition of said second amplification product with the proviso that sequencing of said second amplification product is not performed to determine said base composition.

11. The method of claim 10 wherein said base composition of said second amplification product is determined by mass spectrometry.

12. The method of claim 9 further comprising determining the sequence of said second amplification product.

13. The method of claim 1 wherein said polysorbate 40 is 1% polysorbate 40 (w/v).

14. The method of claim 1 wherein said reaction mixture further comprises dimethyl sulfoxide (DMSO).

15. The method of claim 14 wherein said dimethyl sulfoxide (DMSO) is 2.5% dimethyl sulfoxide (DMSO).

16. The method of claim 1 wherein bovine serum albumin (BSA) is not included in said reaction mixture.

Assignees

• Eshoo Mark W
• Picuri John
• Phillipson Curtis
• Ibis Biosciences Inc

Inventors

• Eshoo Mark W
• Picuri John
• Phillipson Curtis

Classifications

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title