Detection of recombinase polymerase amplification products
First Claim
Patent Images
1. A process of detecting the presence or absence of a recombinase polymerase amplification amplified target nucleic acid, 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 each of said nucleic acid primers comprise a single stranded region at its 3′
end, 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 primers to a sample suspected to contain an amplified target nucleic acid amplified by a recombinase polymerase amplification (RPA) process to form complexes with a first and a second nucleoprotein primer/target nucleic acid structure at a first portion and a second portion of said amplified target nucleic acid, respectively, and wherein said recombinase polymerase amplification process is performed in the presence of a crowding agent such that the crowding agent stimulates amplification;
(c) contacting said 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 target nucleic acid.
12 Assignments
0 Petitions
Accused Products
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.
-
Citations
17 Claims
-
1. A process of detecting the presence or absence of a recombinase polymerase amplification amplified target nucleic acid, 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 each of said nucleic acid primers comprise a single stranded region at its 3′
end, 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 primers to a sample suspected to contain an amplified target nucleic acid amplified by a recombinase polymerase amplification (RPA) process to form complexes with a first and a second nucleoprotein primer/target nucleic acid structure at a first portion and a second portion of said amplified target nucleic acid, respectively, and wherein said recombinase polymerase amplification process is performed in the presence of a crowding agent such that the crowding agent stimulates amplification; (c) contacting said 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 target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
-
Specification