Tagmentation using immobilized transposomes with linkers

Patent metadata

Field	Value
Publication number	US-10920219-B2
Application number	US-201815900717-A
Country	US
Kind code	B2
Filing date	Feb 20, 2018
Priority date	Feb 21, 2017
Publication date	Feb 16, 2021
Grant date	Feb 16, 2021

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:
(i) a transposase,
(ii) a first transposon comprising:
(a) a 3′ portion comprising a first transposon end sequence; and
(b) a first adaptor sequence at the 5′ end of the first transposon end sequence;

(iii) a second transposon comprising a second transposon end sequence complementary to at least a portion of the first transposon end sequence; and
(iv) a non-nucleic acid linker having 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 linker and affinity element have the structure of Formula (I):

wherein:
AE is the affinity element;
Y is C 2-6 alkylene;
X 1 is O, NW, or S;
wherein R 1 is H or C 1-10 alkyl;

n is an integer from 1 to 6;
X 2 is O, CH 2 , or S;
R a is H or —OH; and
Z is absent when R a is H, or is CH 2 when R a is H or OH;
wherein the marks the connection point to the second transposon.

3. The complex of claim 2 , wherein the phosphate group in Formula (I) is connected to a 3′ hydroxyl of the terminal nucleotide of the second transposon.

4. The complex of claim 2 , wherein AE comprises or is an optionally substituted biotin or an amino group.

5. The complex of claim 2 , wherein the linker and affinity element have the structure of Formula (I′):

wherein Z is absent or is CH 2 .

6. The complex of claim 2 , wherein the linker and affinity element have the structure of Formula (Ia):

7. The complex of claim 2 , wherein the linker and affinity element have the structure of Formula (Ib) or (Ic):

where n is 1 or 2;
X 2 is O or CH 2 ; and
Z is absent or is CH 2 .

8. The complex of claim 2 , wherein the linker and affinity element have a structure selected from the group consisting of:

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

10. The complex of claim 9 , wherein the first transposon end sequence and the second transposon end sequence are ME and ME′.

11. The complex of claim 1 , wherein the first adaptor sequence comprises a primer sequence.

12. A first complex according to claim 11 , wherein the first adaptor sequence comprises a first primer sequence, in a mixture comprising a second complex according to claim 11 , wherein the first adaptor sequence of the second complex comprises a second primer sequence.

13. The complexes according to claim 12 , wherein the first primer sequence comprises A14 and the second primer sequence comprises B15.

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

15. The complex of claim 14 , wherein the affinity element is biotin and the affinity binding partner is streptavidin.

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

• C12Q2525/191

  incorporating an adaptor · CPC title