Immobilized transposase complexes for DNA fragmentation and tagging

Patent metadata

Field	Value
Publication number	US-9644199-B2
Application number	US-201313960837-A
Country	US
Kind code	B2
Filing date	Aug 7, 2013
Priority date	Oct 1, 2012
Publication date	May 9, 2017
Grant date	May 9, 2017

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 solid substrate-bound transposase complex, 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. The complex of claim 1 , wherein the transposase component comprises two or more different transposases.

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

Assignees

• Agilent Technologies Inc

Inventors

• Belyaev Alexander S

Classifications

• C12Y207/07

  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

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N11/06 Primary

  attached to the carrier via a bridging agent · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title