Recombinase polymerase amplification

• US 20080076160A1
• Filed: 08/13/2007
• Published: 03/27/2008
• 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, Stemple, Derek
• Granted Patent: US 7,485,428 B2
• Time in Patent Office: Days
• US Class Current: 435/91.1
• 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

1-72. -72. (canceled)

73. A method of recombinase-polymerase amplification of a target sequence polymer molecule, comprising:

(a) contacting a recombinase agent with a first primer sequence molecule to form a first recombinase agent-primer pair and contacting a recombinase agent with a second primer sequence molecule to form a second recombinase agent-primer pair;

(b) contacting the first recombinase agent-primer pair with the target sequence polymer molecule to form a structure, wherein the first primer sequence molecule of the first recombinase agent-primer pair associates with the target sequence polymer molecule;

(c) extending the first primer sequence molecule along the target sequence polymer molecule with a polymerase molecule and monomers to be incorporated into the extended first primer sequence molecule, using the target sequence polymer molecule as a template;

(d) contacting the second recombinase agent-primer pair with the resulting molecule of step (c) to form a structure, wherein the second primer sequence molecule of the second recombinase agent-primer pair associates with the extended first primer sequence molecule;

(e) extending the second primer sequence molecule along the target sequence polymer molecule with a polymerase molecule and monomers to be incorporated into the extended second primer sequence molecule, using the extended first primer sequence molecule as a template; and

(f) repeating steps (b) though (e) until a desired degree of amplification is achieved, wherein the polymerase molecule is capable of displacing the recombinase agent during extension.

76. The method of claim 73, 74, or 75, further comprising:

(a) contacting one or more additional primer sequence molecules with a recombinase agent to form one or more recombinase agent-primer pairs;

(b) contacting the one or more additional recombinase agent-primer pairs with the target sequence polymer molecule or extended primer sequence molecule;

(c) extending the one or more additional primer sequence molecules along the target sequence polymer molecule or extended primer sequence molecule with a polymerase molecule and monomers to be incorporated into the one or more extended primer sequence molecules, using the target sequence polymer molecule or extended primer sequence molecule as a template, wherein the extended one or more additional primer sequence is contained within the first or second extended primer sequence; and

(d) repeating steps (b) and (c) until a desired degree of amplification is achieved.

77. The method of claim 73, 74, or 75, wherein the target sequence polymer molecule is non-natural.

78. The method of claim 73, 74, or 75, wherein the polymerase molecule is PolV.

79. The method of claim 73, 74, or 75, wherein the target sequence molecule is DNA or RNA.

80. The method of claim 73, 74, or 75, wherein one or more primer sequence molecules is associated with a detectable label.

81. The method of claim 73, 74, or 75, further comprising the presence of RecF, RecG, RecO, RecT, RuvA, RuvB, or SSB during extension.

82. The method of claim 73, 74, or 75, wherein the recombinase agent is RecA.

74. A method of recombinase-polymerase amplification of a double-stranded target sequence polymer molecule, comprising:

(a) contacting a recombinase agent with a first primer sequence molecule to form a first recombinase agent-primer pair and contacting a recombinase agent with a second primer sequence molecule to form a second recombinase agent-primer pair;

(b) contacting the first and second recombinase agent-primer pairs with the double-stranded target sequence polymer molecule to form a structure, wherein the first and second primer sequence molecules of the recombinase agent-primer pairs associate with the double-stranded target sequence polymer molecule;

(c) extending the first and second primer sequence molecules along the double-stranded target sequence polymer molecule with a polymerase molecule and monomers to be incorporated into the extended first and second primer sequence molecules, using the double-stranded target sequence polymer molecule as a template; and

(d) repeating steps (b) and (c) until a desired degree of amplification is achieved, wherein the polymerase molecule is capable of displacing the recombinase agent during extension.

75. A method of recombinase-polymerase amplification of a double-stranded target sequence polymer molecule, comprising:

(a) contacting a recombinase agent with a first primer sequence molecule to form a first recombinase agent-primer pair and contacting a recombinase agent with a second primer sequence molecule to form a second recombinase agent-primer pair;

(b) contacting the first and second recombinase agent-primer pairs with the double-stranded target sequence polymer molecule to form a structure, wherein the first and second primer sequence molecules of the recombinase agent-primer pairs associate with the double-stranded target sequence polymer molecule;

(c) extending the first and second primer sequence molecules along the double-stranded target sequence polymer molecule with a polymerase molecule and monomers to be incorporated into the extended first and second primer sequence molecules, using the double-stranded target sequence polymer molecule as a template;

(d) contacting the double-stranded target sequence polymer molecule with primosome assembly proteins, a clamp loader/polymerase III holoenzyme complex, a DNA polymerase III core, a DNA polymerase I, a primase, a helicase, and a ligase;

(e) replicating the strand of the double-stranded target sequence polymer molecule that is displaced by the recombinase agent-primer pair structures through lagging strand synthesis; and

(f) repeating steps (b) through (e) until a desired degree of amplification is achieved.

83. A recombinase-polymerase amplification mixture of substantially purified components, comprising a target sequence polymer molecule, a recombinase agent, a primer sequence molecule, a polymerase molecule, wherein the polymerase molecule is capable of displacing the recombinase agent during an extension reaction.

86. The mixture of claim 83, 84, or 85, wherein the polymerase molecule is PolV.

87. The mixture of claim 83 or 85, wherein the target sequence molecule is DNA or RNA.

88. The mixture of claim 83, 84, or 85, wherein a primer sequence molecule is associated with a detectable label.

89. The mixture of claim 83, 84, or 85, further comprising the presence of RecF, RecG, RecO, RecT, RuvA, RuvB, or SSB during extension.

90. The mixture of claim 83, 84, or 85, wherein the recombinase agent is RecA.

84. A recombinase-polymerase amplification mixture of substantially purified components, comprising a non-natural target sequence polymer molecule, a recombinase agent, a primer sequence molecule, and a polymerase molecule.

85. A recombinase-polymerase amplification mixture of substantially purified components, comprising a target sequence polymer molecule, a recombinase agent, a primer sequence molecule, a polymerase molecule, primosome assembly proteins, a clamp loader/polymerase III holoenzyme complex, a DNA polymerase III core, a DNA polymerase I, a primase, a helicase, and a ligase.