MULTIPLE DISPLACEMENT AMPLIFICATION

• US 20110118151A1
• Filed: 10/15/2010
• Published: 05/19/2011
• Est. Priority Date: 10/15/2009
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Phillipson, Curtis, Picuri, John, Eshoo, Mark W.
• Granted Patent: US 9,890,408 B2
• Time in Patent Office: Days
• US Class Current: 506/39
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/159 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

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.

Claims

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 emulsion-forming components; and

subjecting said reaction mixture to conditions under which said nucleic acid sequence is amplified to produce an amplified product in a multiple displacement reaction.

2. The method of claim 1 wherein said emulsion-forming components comprise a non-ionic detergent and a hydrophobic polymer.

3. The method of claim 1 wherein said non-ionic detergent is selected from the group consisting of Tween 20, Tween 40, Tween 80, Triton X 100 and Triton X 102.

4. The method of claim 3 wherein said non-ionic detergent is present in said reaction mixture at a level between about 0.05% to about 3% (w/v).

5. The method of claim 4 wherein said non-ionic detergent is Tween 40.

6. The method of claim 5 wherein said reaction mixture further comprises betaine, trehalose or both betaine and trehalose.

7. The method of claim 6 wherein said betaine is present in said reaction mixture at a concentration between about 0.2 M to about 1.6 M.

8. The method of claim 6 wherein said trehalose is present in said reaction mixture at a concentration between about 0.1 M and 1.0 M.

9. The method of claim 1 wherein said polymerases comprise 5′

→

3′

DNA polymerase activity, 3′

→

5′

exonuclease activity, and/or 5′

→

3′

excision repair activity.

10. The method of claim 8 wherein said polymerases comprise Phi29 polymerase, Bst DNA polymerase, and/or Pol I polymerase.

11. The method of claim 10 wherein said reaction mixture further comprises between about 0.2 units/μ

L to about 0.6 units/μ

L of Phi29 polymerase.

12. The method of claim 10 wherein said reaction mixture further comprises between about 0.005 units/μ

L to about 0.015 units/μ

L of Pol I polymerase.

13. The method of claim 10 wherein said emulsion-forming components comprise a non-ionic detergent and an oil.

14. The method of claim 2 wherein said hydrophobic polymer comprises polydimethylsiloxane.

15. The method of claim 5 further comprising the step of addition of an emulsion-breaking compound to said reaction to convert said emulsion to aqueous and hydrophobic phases upon completion of said multiple displacement amplification reaction.

16. The method of claim 15 wherein said emulsion-breaking compound is chloroform.

17. The method of claim 15 further comprising amplifying a targeted segment of said amplified product to obtain a second amplification product.

18. The method of claim 17 further comprising determining the base composition of said second amplification product with the provisio that sequencing of said second amplification product is not performed to determine said base composition.

19. The method of claim 18 wherein said base composition of said second amplification product is determined by mass spectrometry.

20. The method of claim 17 further comprising determining the sequence of said second amplification product.

21. A kit for performing multiple displacement amplification of a nucleic acid, said kit comprising:

one or more polymerases;

a plurality of primers; and

an emulsion-forming component.

22. A system for analyzing nucleic acids comprising:

a thermal cycler;

a container for holding a reaction mixture, said container configured for insertion into said thermal cycler, said container compatible with one or more detergents or hydrophobic solvents; and

one or more liquid handlers configured to deliver a nucleic acid sample to said container; and

to deliver an emulsion-breaking compound to said container.

23. The system of claim 22 further comprising a downstream nucleic acid analysis apparatus, configured to receive amplified nucleic acid from said container, said apparatus comprising:

a second thermal cycler configured to perform a targeted second amplification of amplified nucleic acid produced by said system;

a purification unit for purifying a product of said targeted second amplification; and

a nucleic acid analysis unit for determining the base composition or sequence of said targeted second amplification product.

24. The system of claim 23 wherein said purification unit comprises an anion exchange resin linked to a magnetic bead.

25. The system of claim 23 wherein said nucleic acid analysis unit comprises a mass spectrometer or a nucleic acid sequencer.

26. The system of claim 23 further comprising a database of known molecular masses and/or known base compositions of amplification products of known bioagents;

and a controller operably connected to said mass spectrometer and to said database said controller configured to match said molecular masses of said amplification product with a measured or calculated molecular mass of a corresponding amplification product of a bioagent.