Asymmetric PCR with nuclease-free polymerase or nuclease-resistant molecular beacons
First Claim
1. A method for performing combined amplification and detection of a nucleic acid target, the method comprising:
- providing a molecular beacon comprising a region of complementarity to a first region of a first strand of the nucleic acid target;
providing a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target;
providing a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′
of the first region, and the first region being 3′
of the second region;
wherein the first primer is provided at a concentration that is at least about 1.3 times that of the second primer;
providing a template nucleic acid comprising the first strand of the nucleic acid target, a second strand of the nucleic acid target that is complementary to the first strand, or both;
providing a polymerase substantially lacking 5′
to 3′
nuclease activity;
amplifying the target nucleic acid by subjecting the template nucleic acid, the first and second primers, the molecular beacon, and the polymerase to cycles comprising denaturation, annealing, and extension steps; and
, detecting a signal from the molecular beacon at at least one time point during or after the cycles.
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Abstract
This invention provides methods for performing combined asymmetric amplification (e.g., asymmetric PCR amplification) and detection of nucleic acid targets using molecular beacons to detect the products. Methods using a polymerase having reduced or eliminated 5′ to 3′ nuclease activity are provided, as are methods using nuclease-resistant molecular beacons. Asymmetric amplifications using nuclease-free polymerase or nuclease-resistant molecular beacons provide dramatic improvements in signal intensity detected as a result of molecular beacon binding to a target nucleic acid, e.g., during asymmetric PCR. Attendant compositions, systems, devices and kits are also features of the invention.
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Citations
61 Claims
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1. A method for performing combined amplification and detection of a nucleic acid target, the method comprising:
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providing a molecular beacon comprising a region of complementarity to a first region of a first strand of the nucleic acid target;
providing a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target;
providing a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′
of the first region, and the first region being 3′
of the second region;
wherein the first primer is provided at a concentration that is at least about 1.3 times that of the second primer;
providing a template nucleic acid comprising the first strand of the nucleic acid target, a second strand of the nucleic acid target that is complementary to the first strand, or both;
providing a polymerase substantially lacking 5′
to 3′
nuclease activity;
amplifying the target nucleic acid by subjecting the template nucleic acid, the first and second primers, the molecular beacon, and the polymerase to cycles comprising denaturation, annealing, and extension steps; and
,detecting a signal from the molecular beacon at at least one time point during or after the cycles. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for performing combined amplification and detection of a nucleic acid target, the method comprising:
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providing a molecular beacon comprising a region of complementarity to a first region of a first strand of the nucleic acid target, the molecular beacon being resistant to 5′
to 3′
nuclease activity;
providing a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target;
providing a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′
of the first region, and the first region being 3′
of the second region;
wherein the first primer is provided at a concentration that is at least about 1.3 times that of the second primer;
providing a template nucleic acid comprising the first strand of the nucleic acid target, a second strand of the nucleic acid target that is complementary to the first strand, or both;
providing a polymerase;
amplifying the target nucleic acid by subjecting the template nucleic acid, the first and second primers, the molecular beacon, and the polymerase to cycles comprising denaturation, annealing, and extension steps; and
,detecting a signal from the molecular beacon at at least one time point during or after the cycles. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A composition comprising:
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a molecular beacon comprising a region of complementarity to a first region of a first strand of a nucleic acid target;
a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target;
a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′
of the first region, and the first region being 3′
of the second region, wherein the first primer is present at a concentration that is at least about 1.3 times that of the second primer; and
,a polymerase substantially lacking 5′
to 3′
nuclease activity. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A composition comprising:
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a molecular beacon comprising a region of complementarity to a first region of a first strand of a nucleic acid target, the molecular beacon being resistant to 5′
to 3′
nuclease activity;
a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target; and
,a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′
of the first region, and the first region being 3′
of the second region;
wherein the first primer is present at a concentration that is at least about 1.3 times that of the second primer. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)
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Specification