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US 10,767,208 B2Granted
Closed nucleic acid structures
AssigneeGen-Probe Incorporated
InventorsNorman C. Nelson, Jijumon Chelliserry, Steven T. Brentano, Dmitry Lyakhov, Matthew C. Friedenberg, Anne-Laure Shapiro
Life of this patent
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Abstract
The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.
Claims20
A method of forming a closed nucleic acid structure comprising a segment of a target nucleic acid, the method comprising:
- (a) contacting the target nucleic acid with a primer pair under amplification conditions, each of the primers of the pair including a 5′ phosphate group and a 3′ segment, the 3′ segments of the primers being target-binding segments;
- thereby forming an amplified nucleic acid comprising duplex target nucleic acid flanked by the primers of the pair duplexed with their complementary segments, wherein each strand of the amplified nucleic acid includes a 5′ phosphate group and a 3′ hydroxyl group;
- (b) denaturing the amplified nucleic acid and contacting a strand of the denatured amplified nucleic acid with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment, a 3′ segment having a stem-loop structure, and a 3′ hydroxyl group, wherein the 5′ segment is complementary to a segment at the 5′ end of the amplified nucleic acid strand;
- (c) annealing the 5′ segment of the stem-loop adaptor to the 5′ end segment of the amplified nucleic acid strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group of the amplified nucleic acid strand is separated by a nick from the 3′ hydroxyl group of the stem-loop adaptor;
- and
- (d) providing a ligase that seals the nick between the 5′ phosphate group of the amplified nucleic acid strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the amplified nucleic acid strand, thereby forming a closed nucleic acid structure.
3 more elements
A method of forming a closed nucleic acid structure comprising a segment of a target nucleic acid, the method comprising:
- (a) contacting the target nucleic acid with a primer having a 5′ phosphate group and a 3′ segment, the 3′ segment being a target-binding segment;
- (b) annealing the 3′ segment of the primer to the target nucleic acid, thereby forming a template for extension from the primer;
- (c) forming an extended nucleic acid strand duplexed to the target nucleic acid, the extended strand comprising from 5′-3′ the primer and a segment complementary to the target nucleic acid, the 5′ end of the extended strand being the phosphorylated 5′ end of the primer and the 3′ end of the extended strand having a 3′ hydroxyl group;
- (d) denaturing the extended strand from the template nucleic acid and contacting the extended strand with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment, a 3′ segment having a stem-loop structure, and a 3′ hydroxyl group, wherein the 5′ segment is complementary to a segment at the 5′ end of the extended strand;
- (e) annealing the 5′ segment of the stem-loop adaptor to the 5′ segment of the extended strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group of the extended strand is separated by a nick from the 3′ hydroxyl group of the stem-loop adaptor;
- and
- (f) providing a ligase that seals the nick between the 5′ phosphate group of the extended strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the extended strand, thereby forming a closed nucleic acid structure.
4 more elements
A method of forming a closed nucleic acid structure comprising a segment of a target nucleic acid, the method comprising:
- (a) contacting the target nucleic acid with a primer having a 5′ phosphate group and a 3′ segment, the 3′ segment being a target-binding segment;
- (b) annealing the 3′ segment of the primer to the target nucleic acid, thereby forming a template for extension from the primer;
- (c) forming an extended nucleic acid strand duplexed to the target nucleic acid, the extended strand comprising from 5′-3′ the primer and a strand complementary to the target nucleic acid, the 5′ end of the extended strand being the phosphorylated 5′ end of the primer and the 3′ end of the extended strand having a 3′ hydroxyl group;
- (d) denaturing the extended strand from the target nucleic acid and contacting the extended strand with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment having a stem-loop structure, a 3′ segment, and a 3′ hydroxyl group, wherein the 3′ segment is complementary to a segment located at the 3′ end of the extended strand;
- (e) annealing the 3′ segment of the stem-loop adaptor to the 3′ end segment of the extended strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group stem-loop adaptor is separated by a nick from the 3′ hydroxyl group of the extended strand;
- and
- (f) providing a ligase that seals the nick between the 5′ phosphate group of the extended strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the extended strand, thereby forming a closed nucleic acid structure.
4 more elements
Dependencies are inferred from the claim text.