Recombinase polymerase amplification
First Claim
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1. A recombinase polymerase amplification process of amplification of a double stranded target nucleic acid molecule, comprising the steps of:
- (a) contacting UvsX, UvsY, and gp32 proteins with a first and a second single stranded nucleic acid primer specific for said double stranded target nucleic acid molecule to form a first and a second nucleoprotein primer, wherein said UvsX, UvsY, and gp32 are each derived from a myoviridae phage, and wherein no more than two of said UvsX, UvsY and gp32 proteins are T4 phage proteins;
(b) contacting the first nucleoprotein primer to said double stranded target nucleic acid molecule to create a first D loop structure at a first portion of said double stranded target nucleic acid molecule and contacting the second nucleoprotein primer to said double stranded target nucleic acid molecule to create a second D loop structure at a second portion of said double stranded target nucleic acid molecule 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 double stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule;
(c) extending the 3′
end of said first and second nucleoprotein primer with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a first and second double stranded target nucleic acid molecule and a first and second displaced strand of nucleic acid; and
(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached.
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Abstract
The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.
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Citations
22 Claims
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1. A recombinase polymerase amplification process of amplification of a double stranded target nucleic acid molecule, comprising the steps of:
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(a) contacting UvsX, UvsY, and gp32 proteins with a first and a second single stranded nucleic acid primer specific for said double stranded target nucleic acid molecule to form a first and a second nucleoprotein primer, wherein said UvsX, UvsY, and gp32 are each derived from a myoviridae phage, and wherein no more than two of said UvsX, UvsY and gp32 proteins are T4 phage proteins; (b) contacting the first nucleoprotein primer to said double stranded target nucleic acid molecule to create a first D loop structure at a first portion of said double stranded target nucleic acid molecule and contacting the second nucleoprotein primer to said double stranded target nucleic acid molecule to create a second D loop structure at a second portion of said double stranded target nucleic acid molecule 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 double stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule;(c) extending the 3′
end of said first and second nucleoprotein primer with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a first and second double stranded target nucleic acid molecule and a first and second displaced strand of nucleic acid; and(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22)
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19. A recombinase polymerase amplification process of amplification of a double stranded target nucleic acid molecule, comprising the steps of
(a) contacting UvsX and gp32 proteins with a first and a second single stranded nucleic acid primer specific for said double stranded target nucleic acid molecule to form a first and a second nucleoprotein primer, wherein said UvsX and gp32 are each derived from a myoviridae phage, and wherein no more than one of said UvsX and gp32 proteins are T4 phage proteins; -
(b) contacting the first nucleoprotein primer to said double stranded target nucleic acid molecule to create a first D loop structure at a first portion of said double stranded target nucleic acid molecule and contacting the second nucleoprotein primer to said double stranded target nucleic acid molecule to create a second D loop structure at a second portion of said double stranded target nucleic acid molecule 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 double stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule;(c) extending the 3′
end of said first and second nucleoprotein primer with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a first and second double stranded target nucleic acid molecule and a first and second displaced strand of nucleic acid; and(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached, wherein said process is performed in the absence of UvsY.
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21. A recombinase polymerase amplification process of amplification of a double stranded target nucleic acid molecule with a first and a second strand of DNA, comprising the steps of:
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(a) contacting UvsX, UvsY, and gp32 proteins with a first single stranded nucleic acid primer specific for said double stranded target nucleic acid molecule to form a population of first nucleoprotein primer, wherein said UvsX, UvsY, and gp32 are each derived from a myoviridae phage, and wherein no more than two of said UvsX, UvsY and gp32 proteins are T4 phage proteins; (b) contacting the first nucleoprotein primer with said double stranded target nucleic acid molecule thereby forming a first D loop structure at a first portion of said double stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule; (c) extending the 3′
end of said first nucleoprotein primer with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a double stranded target nucleic acid molecule and a displaced strand of nucleic acid molecule;(d) hybridizing a second single stranded nucleic acid primer with said displaced strand of nucleic acid molecule to form a hybridized second single stranded nucleic acid primer; (e) elongating said hybridized second single stranded nucleic acid primer to generate a double stranded target nucleic acid molecule; (f) continuing the reaction through repetition of (b) and (e) until a desired degree of amplification is reached.
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Specification