Selective nucleic acid amplification from nucleic acid pools

Patent metadata

Field	Value
Publication number	US-9862994-B2
Application number	US-201314376241-A
Country	US
Kind code	B2
Filing date	Feb 8, 2013
Priority date	Feb 9, 2012
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

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.

First claim

What is claimed is:

1. 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.

2. The composition of claim 1 , wherein when the 5′ end and the 3′ end are juxtaposed, they are not covalently linked.

3. 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.

4. The composition of claim 1 , wherein the subpool-specific sequence is about 8 to about 20 nucleotides in length.

5. The composition of claim 1 , wherein the endonuclease site is unique.

6. 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.

7. The composition of claim 1 , wherein the primer is hybridized to the subpool-specific sequence.

8. 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.

9. The composition of claim 8 , wherein each subpool comprises 1 to 1000 different target sequences.

10. The composition of claim 9 , wherein the target sequences are single-stranded DNA strands.

11. 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.

12. The method of claim 11 further comprising:
contacting the second plurality of concatemers with a third primer that is complementary to and hybridizes to the subpool-specific sequence and digesting the second plurality of concatemers with an endonuclease to produce a second plurality of monomers;
contacting the second plurality of monomers with ligase in the presence of the third primer; and
amplifying the second plurality of monomers from the third primer via rolling circle amplification to produce a third plurality of concatemers.

13. The method of claim 11 , wherein when the 5′ end and the 3′ end are juxtaposed, they are not covalently linked.

14. The method of claim 11 , wherein the subpool-specific sequence is about 8 to about 20 nucleotides in length.

15. The method of claim 11 , wherein the endonuclease site is unique.

16. The method of claim 11 , wherein the nicking sites are the same.

Assignees

• Dana Farber Cancer Inst Inc

Inventors

• Schmidt Thorsten Lars
• Theilmann Mark
• Shih William M

Classifications

• C12Q2563/155

  Particles of a defined size, e.g. nanoparticles · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q2525/131

  incorporating a restriction site · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• C12Q2563/157

  Nanotubes or nanorods · CPC title