Recombinase polymerase amplification

• US 20090325165A1
• Filed: 01/30/2009
• Published: 12/31/2009
• Est. Priority Date: 02/21/2002
• Status: Abandoned Application

• Current Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: TwistDx, Inc.
• Inventors: Armes, Niall A., Stemple, Derek L.
• Application Number:
  US12/322,354
• Publication Number:
  US 20090325165A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 1/6844 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 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 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: G06F 16/951 Indexing; Web crawling techniques
• CPC Class Codes: G06F 16/9574 of access to content, e.g. by caching

Abstract

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

Claims

1. A RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA, comprising the steps of:

(a) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;

(b) contacting the first and second nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said first strand and form a double stranded structure at a second portion of said second strand such that the 3′

ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the same template nucleic acid molecule;

(c) extending the 3′

end of said first and second nucleoprotein primer with one or more polymerases and dNTPs to generate a first and second double stranded nucleic acid and a first and second displaced strand of nucleic acid

(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached.

2-72. -72. (canceled)