Recombinase polymerase amplification

• US 8,426,134 B2
• Filed: 08/18/2011
• Issued: 04/23/2013
• Est. Priority Date: 02/21/2002
• Status: Expired due to Term

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Inventors: Piepenburg, Olaf, Williams, Colin H., Armes, Niall A., Stemple, Derek L.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US13/212,361
• Publication Number:
  US 20120058517A1
• Time in Patent Office: 614 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

Claims

1. A recombinase polymerase amplification process of DNA amplification of a double stranded target nucleic acid molecule comprising a first strand of DNA and second strand of DNA, the process comprising:

(a) contacting in a solution a uvsX recombinase agent with a first nucleic acid primer and a second nucleic acid primer to form a first nucleoprotein primer and a second nucleoprotein primer, wherein each of the first and second nucleic acid primers comprise a single stranded region at its 3′

end;

(b) contacting the first and the second nucleoprotein primers with the double stranded target nucleic acid molecule thereby forming;

(1) a first double-stranded structure at a first portion of the first strand and

(2) a second double stranded structure at a second portion of the second strand such that the 3′

end of each of the first nucleoprotein primer and the second nucleoprotein primer are oriented toward one another on the same double-stranded template nucleic acid molecule;

(c) extending the 3′

end of each of the first and second nucleoprotein primers with dNTPs and one or more DNA polymerases with strand-displacing properties to generate a first double-stranded nucleic acid product and a second double-stranded nucleic acid product and a first displaced strand of nucleic acid product and a second displaced strand of nucleic acid product; and

(d) repeating (b) and (c) to amplify the double stranded target nucleic acid molecule;

wherein the solution further comprises a gp32 single-stranded DNA binding protein;

wherein the solution further comprises a recombinase loading factor uvsY at a concentration of between 0.2 and 8 micromolar;

wherein the solution further comprises a crowding agent at a concentration of between 1% and 12%;

wherein the one or more DNA polymerase lack 5′

to 3′

exonuclease activity and lack FLAP endonuclease activity;

wherein the solution further comprises a hydrolysable nucleoside triphosphate; and

wherein the process is performed at a temperature of between 20°

C. and 50°

C.

2. A process of nested recombinase polymerase amplification, the process comprising:

(a) amplifying a region of DNA using the recombinase polymerase amplification process of claim 1 using a first primer and a second primer to produce a first amplified product;

(b) amplifying the first amplified product using a third primer and a fourth primer using the recombinase polymerase amplification process of claim 1 to produce a second amplified product, wherein the second amplified product is a smaller sequence contained within the first amplified product.

3. A process comprising:

providing an aqueous solution comprising

(1) at least one recombinase;

(2) at least one single stranded DNA binding protein;

(3) at least one DNA polymerase;

(4) dNTPs or a mixture of dNTPs and ddNTPs;

(5) a reducing agent;

(6) ATP or ATP analog;

(7) at least one recombinase loading protein; and

(8) a crowding agent; and

freeze-drying the aqueous solution.

4. The process of claim 3, wherein the at least one recombinase comprises a uvsX recombinase.

5. The process of claim 3, wherein the at least one single stranded DNA binding protein comprises a gp32 single stranded DNA binding protein.

6. The process of claim 3, wherein the at least one DNA polymerase comprises a DNA polymerase I fragment.

7. The process of claim 3, wherein the reducing agent comprises dithiothreitol (DTT).

8. The process of claim 3, wherein the at least one recombinase loading protein comprises a uvsY recombinase loading protein.

9. The process of claim 3, wherein the aqueous solution further comprises one or more of potassium acetate, magnesium acetate, phosphocreatine, and creatine kinase.

10. The process of claim 3, wherein the crowding agent comprises polyethylene glycol.

11. The process of claim 3, wherein the aqueous solution further comprises at least one nucleic acid primer.

12. The process of claim 3, wherein:

the at least one recombinase comprises a uvsX recombinase;

the at least one single stranded DNA binding protein comprises a gp32 single stranded DNA binding protein;

the at least one DNA polymerase comprises a DNA polymerase I fragment;

the reducing agent comprises dithiothreitol (DTT); and

the at least one recombinase loading protein comprises a uvsY recombinase loading protein.

13. The process of claim 12, wherein the aqueous solution further comprises one or more of potassium acetate, magnesium acetate, phosphocreatine, and creatine kinase.

14. The process of claim 12, wherein the crowding agent comprises polyethylene glycol.

15. The process of claim 3, wherein:

the at least one recombinase comprises a uvsX recombinase;

the at least one single stranded DNA binding protein comprises a gp32 single stranded DNA binding protein;

the at least one DNA polymerase comprises a DNA polymerase I fragment;

the reducing agent comprises dithiothreitol (DTT); and

the at least one recombinase loading protein comprises a uvsY recombinase loading protein;

the crowding agent comprises polyethylene glycol, and

wherein the aqueous solution further comprises potassium acetate, magnesium acetate, phosphocreatine, and creatine kinase.

16. The process of claim 15, wherein the aqueous solution further comprises trehalose.

17. The process of claim 15, wherein the aqueous solution further comprises at least one nucleic acid primer.

18. The process of claim 3, wherein the aqueous solution is freeze-dried onto a solid support.

19. The process of claim 12, wherein the aqueous solution is freeze-dried onto a solid support.

20. The process of claim 15, wherein the aqueous solution is freeze-dried onto a solid support.