Tagmentation using immobilized transposomes with linkers

Patent metadata

Field	Value
Publication number	US-11708573-B2
Application number	US-202117140434-A
Country	US
Kind code	B2
Filing date	Jan 4, 2021
Priority date	Feb 21, 2017
Publication date	Jul 25, 2023
Grant date	Jul 25, 2023

Abstract

The present disclosure relates to methods, compositions, and kits for treating target nucleic acids, including methods and compositions for fragmenting and tagging nucleic acid (e.g., DNA) using transposome complexes bound to a solid support.

First claim

What is claimed is:

1. A transposome complex comprising:
a. a transposase,
b. a first transposon comprising:
i. a 3′ portion comprising a first transposon end sequence; and
ii. a first adaptor sequence at the 5′ end of the first transposon end sequence;
c. a second transposon comprising a second transposon end sequence complementary to at least a portion of the first transposon end sequence; and
d. a cleavable linker comprising one or more photocleavable or enzymatically cleavable nucleotides, wherein the cleavable linker has a first end attached to the 3′ end of the second transposon and a second end attached to an affinity element.

2. The complex of claim 1 , wherein the affinity element is capable of binding to an affinity binding partner on a solid support.

3. The complex of claim 2 , wherein the solid support is a tube, a well of a plate, a slide, a bead, or a flowcell.

4. The complex of claim 3 , wherein the solid support is a paramagnetic bead.

5. The complex of claim 2 , wherein the affinity element is biotin and the affinity binding partner is streptavidin.

6. The complex of claim 1 , wherein the adaptor sequence comprises a universal sequence, primer sequence, or sequencing related sequence.

7. The complex of claim 1 , wherein the transposase is a Tn5 transposase.

8. The complex of claim 7 , wherein the Tn5 transposase is a wild-type Tn5 transposase or a hyperactive Tn5 transposase, or a mutant thereof.

9. The complex of claim 7 , wherein the transposase is conjugated to a purification tag.

10. The complex of claim 7 , wherein the first transposon end sequence and the second transposon end sequence are ME (SEQ ID NO: 8) and ME′ (SEQ ID NO: 5), respectively.

11. The complex of claim 1 , wherein the one or more photocleavable or enzymatically cleavable nucleotides are independently chosen from uracil, uridine, 8-oxo-guanine, xanthine, hypoxanthine, 5,6-dihydrouracil, 5-methylcytosine, thymine-dimer, 7-methylguanosine, 8-oxo-deoxyguanosine, xanthosine, inosine, dihydrouridine, bromodeoxyuridine, uridine, or 5-methylcytidine.

12. The complex of claim 11 , where one or more photocleavable or enzymatically cleavable nucleotides are uracil.

13. The complex of claim 11 , wherein the cleavable linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 photocleavable or enzymatically cleavable nucleotides.

Assignees

• Illumina Inc
• Illumina Cambridge Ltd

Inventors

• Desantis Grace
• Gross Stephen M
• Li jian-sen
• Morrell Natalie
• Slatter Andrew
• Shen Kevin
• Snow Samantha

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N11/06 Primary

  attached to the carrier via a bridging agent · CPC title