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US 8,574,846 B2Granted
Recombinase polymerase amplification
AssigneeAlere San Diego, Inc.
InventorsOlaf Piepenburg, Colin H. Williams, Niall A. Armes, Derek L. Stemple
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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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundres of megabases in length.
Claims15
A process comprising:
- (a) converting a single stranded DNA or RNA molecule into a double stranded nucleic acid molecule comprising a first and second strand of DNA;
- (b) contacting a recombinase agent selected from the group consisting of uvsX and recA with a first and second nucleic acid primer to form a first and second nucleoprotein primer;
- (c) contacting the first and the second nucleoprotein primer to said double stranded nucleic acid molecule thereby forming a first double-stranded structure at a first portion of said first strand and forming a second 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 one another on the same double-stranded template nucleic acid molecule;
- (d) extending the 3′ ends of said first and second nucleic acid primer with one or more polymerases and dNTPs to generate a first and second double-stranded nucleic acid and first and second displaced strands of nucleic acid;
- and
- (e) continuing the reaction through repetition of (c) and (d) until a desired degree of amplification is reached, wherein said process at steps (c) and (d) is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.
3 more elements
Dependencies are inferred from the claim text.