IP Library Granted Patent US 9,309,502
Granted Patent B2
US 9,309,502 · App. 14/610,915 · Granted Apr 12, 2016

Recombinase polymerase amplification

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Quick Facts
Patent No.
US 9,309,502
App. No.
14/610,915
Granted
Apr 12, 2016
Kind
B2
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 (12)

1. A composition comprising;

a T4 bacteriophage UvsX;

a T4 bacteriophage single-stranded DNA binding protein (gp32);

a DNA polymerase; and

a crowding agent.

2. The composition of claim 1 , wherein the DNA polymerase is selected from the group consisting of E. coli DNA polymerase I Klenow fragment, B. stearothermophilus polymerase (Bst), B. subtilis Phi-29 polymerase, and B. subtilis polymerase I (Bsu).

3. The composition of claim 1 or 2 , further comprising ATP or an ATP analog, dNTP(s), or T4 bacteriophage UvsY.

4. The composition of claim 1 , wherein the crowding agent is selected from the group consisting of polyethylene glycol (PEG), dextran and Ficoll.

5. The composition of claim 4 , wherein the PEG is PEG1450, PEG3000, PEG8000 or PEG10000.

6. The composition of claim 4 , wherein the PEG has a molecular weight between 15000 and 20000.

7. The composition of claim 3 , wherein the dNTP(s) is/are selected from the group consisting of dATP, dGTP, dCTP and dTTP.

8. The composition of claim 3 , wherein the ATP or ATP analog is selected from the group consisting of ATP, ATP-γ-S, ATB-β-S, ddATP, and a combination thereof.

Assignments (5)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 10, 2020
From: ALERE SAN DIEGO INC.
To: ABBOTT DIAGNOSTICS SCARBOROUGH, INC.
Reel/Frame 054604/0936 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 2, 2015
From: PIEPENBURG, OLAF; WILLIAMS, COLIN H.; ARMES, NIALL A.; STEMPLE, DEREK L.
To: ASM SCIENTIFIC, INC.
Reel/Frame 034863/0038 →
CHANGE OF NAME Recorded Feb 2, 2015
From: ASM SCIENTIFIC, INC.
To: TWISTDX, INC.
Reel/Frame 034863/0125 →
MERGER Recorded Feb 2, 2015
From: TWISTDX, INC.
To: BIOSITE INCORPORATED
Reel/Frame 034863/0235 →
CHANGE OF NAME Recorded Feb 2, 2015
From: BIOSITE INCORPORATED
To: ALERE SAN DIEGO, INC.
Reel/Frame 034870/0685 →