Sample preparation on a solid support

Patent metadata

Field	Value
Publication number	US-10041066-B2
Application number	US-201514671071-A
Country	US
Kind code	B2
Filing date	Mar 27, 2015
Priority date	Jan 9, 2013
Publication date	Aug 7, 2018
Grant date	Aug 7, 2018

Abstract

Presented are methods and compositions for using immobilized transposase and a transposon end for generating an immobilized library of 5′-tagged double-stranded target DNA on a surface. The methods are useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including massively parallel DNA sequencing.

First claim

What is claimed is:

1. A solid support having transposome complexes immobilized thereon, wherein said transposome complexes comprise a transposase bound to a first polynucleotide, wherein the first polynucleotide comprises:
(i) a 3′ portion comprising a transposon end sequence, and
(ii) a first tag comprising a first tag domain,
wherein the transposome complexes are homodimers, the homodimers comprising a first plurality of homodimers comprising a first polynucleotide of a first sequence and a second plurality of homodimers comprising a first polynucleotide of a second sequence.

2. The solid support of claim 1 , wherein the tag domain comprises a region for cluster amplification.

3. The solid support of claim 1 , wherein the tag domain comprises a region for priming a sequencing reaction.

4. The solid support of claim 1 , wherein the tag sequence comprises an index domain, an amplification domain, or both.

5. The solid support of claim 1 , wherein the transposome complexes comprise a hyperactive Tn5 transposase.

6. The solid support of claim 1 , wherein the solid support comprises microparticles or beads.

7. The solid support of claim 1 , wherein the solid support comprises a patterned surface or an array of wells.

8. The solid support of claim 1 , wherein the transposome complexes are present on the solid support at a density of at least 103, 104, 105, or 106 complexes per mm 2 .

9. The solid support of claim 1 , wherein the first polynucleotide is immobilized to the solid support.

10. The solid support of claim 9 , wherein the first polynucleotide comprises a cleavage moiety that allows for cleavage of the first polynucleotide from the solid support.

11. The solid support of claim 10 , wherein the cleavage moiety is at the 5′ end of the first polynucleotide.

12. The solid support of claim 1 , wherein the transposome complexes comprise a second polynucleotide comprising a region complementary to the transposon end sequence.

13. The solid support of claim 12 , wherein the second polynucleotide is hybridized to the 3′ end of the first polynucleotide, and the first polynucleotide is immobilized at its 5′ end to the surface of the solid support.

14. The solid support of claim 1 , wherein the transposome complexes comprise a dimer of Tn5 or hyperactive Tn5.

15. The solid support of claim 1 , wherein the first sequence and the second sequence comprise different amplification primer sequences, and wherein the first sequence and the second sequence optionally comprise index tags.

16. A method of fragmenting target DNA, said method comprising: combining the target DNA with the solid support of claim 1 under conditions that permit transposase-mediated cleavage of the target DNA.

17. The method of claim 16 , wherein said cleavage produces solid support-bound, fragmented DNA.

Assignees

• Illumina Cambridge Ltd

Inventors

• Gormley Niall Anthony
• Smith Geoffrey Paul

Classifications

• C12N15/1065 Primary

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

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/00608

  DNA chips · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6834 Primary

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title