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US 9,284,602 B2Granted
Compositions of toehold primer duplexes and methods of use
AssigneePresident and Fellows of Harvard College
InventorsDavid Yu Zhang, Peng Yin
Life of this patent
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Abstract
Provided herein are primers and primer systems having improved specificity and kinetics over existing primers, and methods of use thereof.
Claims20
A partially double-stranded primer comprised of first and second nucleic acid strands arranged into
- (1) one double-stranded target-non-specific region,
- (2) one double-stranded target-specific region, and
- (3) one single-stranded target-specific region contributed to by the first nucleic acid strand,
- wherein the double-stranded target-non-specific region has a standard free energy that is within 10% of the standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of the first nucleic acid strand that contributes to region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
1 more element
A system comprising
- a nucleic acid target,
- a polymerase, and
- a partially double-stranded primer comprising a first and a second nucleic acid strand arranged into
- (1) one double-stranded target-non-specific region,
- (2) one double-stranded target-specific region, and
- (3) one single-stranded target-specific region contributed to by the first nucleic acid strand,
- wherein the double-stranded target-non-specific region has a standard free energy that is within 10% of the standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of the first nucleic acid strand that contributes to region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
4 more elements
A kit comprising
- a first single-stranded nucleic acid in a first container, and
- a second single-stranded nucleic acid that is complementary to a region of the first single-stranded nucleic acid, in a second container,
- wherein, when the first and second single-stranded nucleic acids are hybridized to each other, a partially double-stranded nucleic acid is formed that comprises
- (1) a double-stranded target-non-specific region,
- (2) a double-stranded target-specific region, and
- (3) a single-stranded target-specific region contributed to by the first nucleic acid,
- wherein the double-stranded target-non-specific region has a standard free energy that is within 10% of the standard free energy for the single-stranded target-specific region bound to a perfectly complementary region of a target nucleic acid, wherein the sequence of the first nucleic acid strand that contributes to region (1) is not perfectly complementary to and does not bind to the target nucleic acid, and wherein the sequences of regions (2) and (3) are complementary to and bind to the target nucleic acid.
4 more elements
Dependencies are inferred from the claim text.