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US 9,862,994 B2Granted
Selective nucleic acid amplification from nucleic acid pools
AssigneeDana-Farber Cancer Institute, Inc.
InventorsThorsten Lars Schmidt, Mark Theilmann, William M. Shih
Life of this patent
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Abstract
Provided herein are nucleic acids and methods for selectively amplifying in parallel tens of thousands of high quality oligonucleotides without common sequences. The resultant oligonucleotides can be used for a variety of purposes and applications including but not limited to DNA nano structure synthesis.
Claims16
A composition comprising:
- (a) a single-stranded template nucleic acid comprising
- a target sequence,
- a 5′ end having a first sequence, and
- a 3′ end having a second sequence such that, when the 5′ end and the 3′ end are juxtaposed, the first sequence and the second sequence form a subpool-specific region, wherein the subpool-specific region comprises a subpool-specific sequence and an endonuclease site and is flanked on each end by a nicking site;
- and
- (b) a primer that is complementary to and hybridizes to the subpool-specific sequence, wherein the target sequence is about 15 to about 100 nucleotides in length.
3 more elements
A composition comprising:
- (a) a single-stranded template nucleic acid comprising
- a target sequence,
- a 5′ end having a first sequence, and
- a 3′ end having a second sequence such that, when the 5′ end and the 3′ end are juxtaposed, the first sequence and the second sequence form a subpool-specific region, wherein the subpool-specific region comprises a subpool-specific sequence and an endonuclease site and is flanked on each end by a nicking site;
- and
- (b) a primer that is complementary to and hybridizes to the subpool-specific sequence, wherein the subpool-specific region comprises at least two subpool-specific sequences, each subpool-specific sequence comprising an endonuclease site.
3 more elements
A composition comprising:
- (a) a single-stranded template nucleic acid comprising
- a target sequence,
- a 5′ end having a first sequence, and
- a 3′ end having a second sequence such that, when the 5′ end and the 3′ end are juxtaposed, the first sequence and the second sequence form a subpool-specific region, wherein the subpool-specific region comprises a subpool-specific sequence and an endonuclease site and is flanked on each end by a nicking site;
- and
- (b) a primer that is complementary to and hybridizes to the subpool-specific sequence, wherein the nicking sites are the same.
3 more elements
A composition comprising
- (a) a plurality of single-stranded nucleic acids, each comprising a target sequence,
- a 5′ end having a first sequence, and
- a 3′ end having a second sequence such that, when the 5′ end and the 3′ end are juxtaposed, the first sequence and the second sequence form a subpool-specific region, wherein the subpool-specific region comprises a subpool-specific sequence and an endonuclease site and is flanked on each end by a nicking site;
- and
- (b) a plurality of primers that are complementary to and hybridize to the subpool-specific sequences, wherein the plurality of single-stranded nucleic acids comprises at least two subpools of single-stranded nucleic acids, each subpool having a unique subpool-specific sequence.
2 more elements
A method comprising:
- combining in a reaction mixture (a) a plurality of single-stranded nucleic acids, wherein each single-stranded nucleic acid comprises
- a target sequence,
- a 5′ end having a first sequence, and
- a 3′ end having a second sequence such that, when the 5′ end and the 3′ end are juxtaposed, the first sequence and the second sequence form a subpool-specific region, wherein the subpool-specific region comprises a subpool-specific sequence and an endonuclease site and is flanked on each end by a nicking site, (b) ligase, and (c) a first primer that is complementary to and hybridizes to the subpool-specific sequence;
- amplifying single-stranded nucleic acids using the first primer via rolling circle amplification to produce a first plurality of concatemers;
- contacting the first plurality of concatemers with a second primer that is complementary to and hybridizes to a sequence complementary to the subpool-specific sequence and digesting the first plurality of concatemers with an endonuclease to produce a first plurality of monomers;
- contacting the first plurality of monomers with ligase in the presence of the second primer;
- amplifying the first plurality of monomers from the second primer via rolling circle amplification to produce a second plurality of concatemers.
5 more elements
Dependencies are inferred from the claim text.