Recombinase polymerase amplification

• US 20090017462A1
• Filed: 05/07/2008
• Published: 01/15/2009
• Est. Priority Date: 02/21/2002
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: ASM Scientific, Inc.
• Inventors: Armes, Niall A., Piepenburg, Olaf, Williams, Colin H., Stemple, Derek L.
• Granted Patent: US 7,763,427 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• 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 2527/101 Temperature
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

This disclosure describe three 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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundreds of megabases in length.

Claims

1-98. -98. (canceled)

99. A process of detecting the presence or absence of a recombinase polymerase amplification amplified nucleic acids product, comprising the steps of:

(a) contacting a recombinase agent with a first and second nucleic acid primer to form a first and second nucleoprotein primer, wherein said nucleic acid primer comprises a single stranded region at its 3′

, wherein said first nucleic acid primer is labeled with a first member of a first binding pair, and wherein said second nucleic acid primer is labeled with a first member of a second binding pair;

(b) contacting the first and the second nucleoprotein primer to a sample suspected to contain a recombinase polymerase amplification amplified nucleic acid to form primer/recombinase polymerase amplification amplified nucleic acid complexes;

(c) contacting said primer/recombinase polymerase amplification amplified nucleic acid complexes with a first mobile solid support coated with a second member of said first binding pair and a second mobile solid support coated with a second member of said second binding pair;

(d) determining if said first mobile solid support is co-localized with said second mobile solid support to determine the presence of said recombinase polymerase amplification amplified nucleic acid product.

100. The process of claim 99 wherein the first nucleic acid primer has the same nucleic acid sequence as a primer used to produce the suspected recombinase polymerase amplification amplified nucleic acid product.

101. The process of claim 99 wherein the second nucleic acid primer has the same nucleic acid sequence as a primer used to produce the suspected recombinase polymerase amplification amplified nucleic acid product.

102. The process of claim 99 wherein the first and/or second primers are associated with the target DNA by virtue of the formation of a triple helix mediated by a recombinase, and occurring after amplification of the target by an recombinase polymerase amplification reaction.

103-187. -187. (canceled)