Immobilized transposase complexes for DNA fragmentation and tagging

Patent metadata

Field	Value
Publication number	US-10100348-B2
Application number	US-201615348743-A
Country	US
Kind code	B2
Filing date	Nov 10, 2016
Priority date	Oct 1, 2012
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

The present invention provides a simple and rapid method for preparing purified transposase complexes that are highly suited for fragmenting DNA. The method includes forming transposase complexes with oligonucleotide adapters in cell lysate, then purifying the complexes from the other substance in the cell lysate. Purification is accomplished using a specific binding pair, in which one member of the pair is bound to an oligonucleotide adapter of the complex and the other member of the pair is bound to a solid substrate. The bound complexes can be immediately used in DNA fragmentation reactions to produce solid substrate-bound DNA fragments, which can be used for any number of purposes, including as templates for amplification and sequencing.

First claim

The invention claimed is:

1. A method of fragmenting target DNA, said method comprising:
combining the target DNA with a solid substrate-bound complex under conditions that permit transposase-mediated cleavage of the target DNA,

wherein the complex comprises:
a transposase component comprising a first transposase and a second transposase;
an oligonucleotide adapter component comprising a first-oligonucleotide adapter and a second oligonucleotide adapter,
wherein each oligonucleotide adapter comprises at least one double stranded portion that contains a recognition sequence for a transposase of the complex, and
wherein the first oligonucleotide adapter is bound to the first transposase and the second oligonucleotide adapter is bound to the second transposase;

a linker component comprising a specific binding pair, one of the members of the specific binding pair being bound to the first oligonucleotide adapter and the other member of the specific binding pair being bound to a solid substrate, wherein the second oligonucleotide adapter is not bound to a member of any specific binding pair; and
a solid substrate.

2. A method of producing solid substrate-bound fragmented DNA, said method comprising:
combining target DNA with a solid substrate-bound complex under conditions that permit transposase-mediated cleavage of the target DNA, said cleavage resulting in production of solid substrate-bound fragmented DNA,

wherein the complex comprises:
a transposase component comprising a first transposase and a second transposase;
an oligonucleotide adapter component comprising a first-oligonucleotide adapter and a second oligonucleotide adapter,
wherein each oligonucleotide adapter comprises at least one double stranded portion that contains a recognition sequence for a transposase of the complex, and
wherein the first oligonucleotide adapter is bound to the first transposase and the second oligonucleotide adapter is bound to the second transposase;

a linker component comprising a specific binding pair, one of the members of the specific binding pair being bound to the first oligonucleotide adapter and the other member of the specific binding pair being bound to a solid substrate, wherein the second oligonucleotide adapter is not bound to a member of any specific binding pair; and
a solid substrate.

3. The method of claim 1 , wherein the transposase component comprises two or more different transposases.

4. The method of claim 1 , wherein each adapter further comprises at least one single-stranded portion.

5. The method of claim 1 , wherein the target DNA and the solid substrate-bound complex are in a composition.

6. The method of claim 5 , wherein the composition comprises a nuclease inhibitor.

7. The method of claim 6 , wherein the nuclease inhibitor is a divalent cation chelating agent.

8. The method of claim 5 , wherein the composition comprises a cell lysate.

9. The method of claim 1 , wherein the specific binding pair comprises avidin or streptavidin and biotin, a nano-tag and streptavidin, an antibody or antigen-binding portion thereof and an antigen epitope to which the antibody specifically binds, an enzyme-substrate pair, poly-histidine and a nickel-based resin, an aptamer and their specific target molecules, or an Si-tag and a silica particle.

10. The method of claim 2 , wherein the transposase component comprises two or more different transposases.

11. The method of claim 2 , wherein each adapter further comprises at least one single-stranded portion.

12. The method of claim 2 , wherein the target DNA and the solid substrate-bound complex are in a composition.

13. The method of claim 12 , wherein the composition comprises a nuclease inhibitor.

14. The method of claim 13 , wherein the nuclease inhibitor is a divalent cation chelating agent.

15. The method of claim 12 , wherein the composition comprises a cell lysate.

16. The method of claim 2 , wherein the specific binding pair comprises avidin or streptavidin and biotin, a nano-tag and streptavidin, an antibody or antigen-binding portion thereof and an antigen epitope to which the antibody specifically binds, an enzyme-substrate pair, poly-histidine and a nickel-based resin, an aptamer and their specific target molecules, or an Si-tag and a silica particle.

Assignees

• Agilent Technologies Inc

Inventors

• Belyaev Alexander S

Classifications

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title