Sample preparation on a solid support

• US 10,041,066 B2
• Filed: 03/27/2015
• Issued: 08/07/2018
• Est. Priority Date: 01/09/2013
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Smith, Geoffrey Paul
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/671,071
• Publication Number:
  US 20150284714A1
• Time in Patent Office: 1,229 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

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.

Claims

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 mm².

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.