Arrays and methods of use

• US 9,376,677 B2
• Filed: 01/14/2016
• Issued: 06/28/2016
• Est. Priority Date: 03/16/2001
• Status: Expired due to Term

• Current Assignee: Invitae Corporation
• Original Assignee: Singular Bio, Inc. (Invitae Corporation)
• Inventors: Mir, Kalim
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/995,946
• Publication Number:
  US 20160122754A1
• Time in Patent Office: 166 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/283.1, 435/287.1, 435/287.2, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 422/50, 422/68.1
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00513 Essentially linear supports
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00524 in the shape of fiber bundles
• CPC Class Codes: B01J 2219/00554 Physical means
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• 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: C12Q 1/6818 : involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/303 : Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

Methods are provided for producing a molecular array comprising a plurality of molecules immobilized to a solid substrate at a density which allows individual immobilized molecules to be individually resolved, wherein each individual molecule in the array is spatially addressable and the identity of each molecule is known or determined prior to immobilization. The use of spatially addressable low density molecular arrays in single molecule detection and analysis techniques is also provided. Novel assays and methods are also provided.

Claims

1. A method of producing a molecular array comprising:

(i) preselecting a plurality of oligonucleotides to be immobilised;

(ii) labeling at least a portion of the plurality of oligonucleotides with one or more labels, thereby producing labeled oligonucleotides;

(iii) immobilising at least a portion of the labeled oligonucleotides on a solid support at a density to allow each of said at least a portion of the labeled oligonucleotides on the solid support to be individually resolved, thereby forming two or more separate and discrete elements, at least two elements of said two or more elements being spatially addressable, each of said at least two elements comprising a plurality of labeled immobilised oligonucleotides from said at least a portion of the labeled oligonucleotides,

wherein sequence identities of said at least a portion of the plurality of labeled immobilised oligonucleotides in each of said at least two elements is specified by a location of each of said at least two elements in which the oligonucleotides are contained; and

(iv) analysing whether at least a portion of the labeled immobilised oligonucleotides of said at least two elements is individually optically resolvable from another portion of the labeled immobilised oligonucleotides, whereby said at least a portion of the labeled immobilised oligonucleotides on each of said at least two elements is individually optically resolvable from the another portion of the labeled immobilised oligonucleotides.

2. The method according to claim 1, wherein the one or more labels can be detected by optical methods.

3. The method according to claim 2, wherein the one or more labels is selected from the group consisting of a single optically active dye and fluorescent molecule, and a plurality of optically active dyes and fluorescent molecules.

4. The method according to claim 1, wherein the oligonucleotides are cDNA and/or genomic DNA.

5. The method according to claim 1, wherein the immobilising step comprises applying the labeled oligonucleotides to the solid support by a method selected from the group consisting of printing, electronic addressing, in situ light-directed synthesis, ink jet synthesis, physical masking, and spotting.

6. The method according to claim 1, wherein the plurality of oligonucleotides are identical oligonucleotides.

7. The method according to claim 1, wherein the plurality of oligonucleotides include functional molecules with different identities.

8. The method according to claim 1, wherein the immobilising step comprises centrifuging the labeled oligonucleotides on the solid support.

9. The method according to claim 1, wherein the solid support comprises a material selected from the group consisting of glass, quartz, silicon wafers, mica, ceramics organic polymers, plastics, polystyrene and polymethacrylate.

10. The method according to claim 1, wherein the labeled immobilised oligonucleotides are separated by a distance of approximately at least 250 nm.

11. The method according to claim 1, wherein the labeled immobilised oligonucleotides are separated by a distance of approximately at least 800 nm.

12. The method according to claim 1, wherein the labeled immobilised oligonucleotides are separated by a distance from 250 to 800 nm.

13. The method according to claim 1, wherein one of the at least two elements is adjacent to another element.

14. The method according to claim 1, wherein each of the at least two elements is from 1 μ

m to 300 μ

m apart.

15. The method according to claim 1, wherein each of the at least two elements has a dimension from 150 μ

m to 300 μ

m.

16. The method according to claim 1, wherein each of the at least two elements has a dimension larger than 500 μ

m.

17. The method according to claim 1, wherein the oligonucleotides have a length from 13 to 25 nucleotides.

18. The method according to claim 1, wherein the oligonucleotides have a length longer than 25 nucleotides.

19. The method according to claim 1, wherein the at least two elements are separated by a raised region or an etched trench.

20. The method according to claim 1, wherein the one or more labels are fluorescent labels.

21. The method according to claim 1, further comprising counting the number of the labeled immobilised oligonucleotides in each element of the at least two elements.

22. The method according to claim 21, wherein the labeled immobilised oligonucleotides within each element of the at least two elements are counted for one or more diagnostic purposes.

23. The method according to claim 1, wherein the labeling is performed by hybridising a labeled probe to only a portion of the plurality of oligonucleotides.

24. The method of claim 23, wherein the labeled probe is a labeled oligonucleotide.

25. The method according to claim 1, wherein the one or more labels are two or more different labels.

26. The method according to claim 1, wherein the solid support is a microtiter plate comprising raised region separating the two or more separate and discrete elements.

27. The method according to claim 1, wherein said at least a portion of the labeled immobilised oligonucleotides comprises circularized DNA.

28. The method according to claim 1, wherein at least a portion of the plurality of oligonucleotides is amplified by rolling circle amplification.

29. The method according to claim 1, wherein

said plurality of labeled immobilized oligonucleotides comprise first immobilising means selected from the group consisting of (i) biotins, (ii) sulfhydryl (SH) groups, (iii) amine groups, (iv) phenylboronic acid (PBA) groups, and (v) acrydite groups, and

said solid support comprises second immobilising means selected from the group consisting of (i) avidin, strepatavidin, or neutravidin, (ii) SH groups, (iii) activated carboxylate or aldehyde groups, (iv) salicylhydroxamic acid (SHA) groups, and (v)thiol surface, silane surface, or the surface containing acrylamide monomers.

30. A method of performing an assay comprising:

producing the molecular array according to the method of claim 1, and performing the assay comprising counting a total number of at least a part of the plurality of labeled immobilised oligonucleotides of said at least two elements.