Arrays and methods of use

• US 9,481,883 B2
• Filed: 07/22/2015
• Issued: 11/01/2016
• 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:
  US14/806,096
• Publication Number:
  US 20150337296A1
• Time in Patent Office: 468 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• 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 immoblized 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:

providing a solid support comprising a plurality of physically discrete elements, wherein one element of said plurality of physically discrete elements is separated from another element of said plurality of physically discrete elements by one or more raised regions or trenches and each of said plurality of physically discrete elements is arranged at predetermined locations on said solid support and is separated by a fixed distance; and

immobilizing a plurality of target oligonucleotide molecules onto said plurality of physically discrete elements, thereby producing immobilized target oligonucleotide molecules and a molecular array comprising said plurality of physically discrete elements having said immobilized target oligonucleotide molecules, wherein each of said plurality of physically discrete elements of the molecular array contains only one of said immobilized target oligonucleotide molecules.

2. The method of claim 1, further comprising amplifying said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements and creating multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements, wherein said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements are a single molecule species.

3. The method of claim 2, further comprising labeling at least a portion of said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements with one or more labels, thereby producing labeled said only one of said target oligonucleotide molecules in each of said plurality of physically discrete elements.

4. The method of claim 2, further comprising sequencing at least a portion of said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements.

5. The method of claim 1, further comprising sequencing said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete elements.

6. The method of claim 5, wherein said sequencing said only one of said immobilized target oligonucleotide molecules comprises sequencing by synthesis.

7. The method of claim 1, where the solid support is rigid or semi-rigid.

8. The method of claim 1, wherein said plurality of physically discrete elements comprise a plurality of wells or nanovials.

9. The method of claim 8, wherein each of at least a portion of said plurality of wells has a dimension that is 10 microns or less.

10. The method of claim 8, wherein the solid support is planar except for the locations of the wells.

11. The method of claim 8, wherein said one or more raised regions separate two or more adjacent wells of said plurality of wells.

12. The method of claim 1, wherein each of said plurality of physically discrete elements has a dimension that is 100 microns or less.

13. The method of claim 1, wherein each of said plurality of physically discrete elements has a size that is 0.1 micron×

0.1 micron or above.

14. The method of claim 1, wherein

each of at least a portion of the plurality of physically discrete elements comprises a plurality of intermediate molecules selected from the group consisting of avidin, streptavidin, silane linkers, polyethylene glycol spacers, and polylysine, and

the immobilizing step comprises interacting the plurality of target oligonucleotide molecules with the plurality of intermediate molecules.

15. The method of claim 1, wherein the fixed distance is determined prior to the immobilizing step.

16. A method of producing a molecular array comprising:

providing a solid support comprising a plurality of physically discrete wells, wherein each of said plurality of physically discrete wells is arranged at predetermined locations of said solid support; and

immobilizing a plurality of target oligonucleotide molecules into said physically discrete wells, thereby producing immobilized target oligonucleotide molecules and a molecular array comprising said plurality of physically discrete wells having said immobilized target oligonucleotide molecules, wherein each of said plurality of physically discrete wells of the molecular array contains only one of said immobilized target oligonucleotide molecules.

17. The method of claim 16, further comprising amplifying said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells and creating multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells, wherein said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells are a single molecule species.

18. The method of claim 17, further comprising labeling at least a portion of said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells with one or more labels, thereby producing labeled said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells.

19. The method of claim 17 further comprises sequencing said only one of said immobilized target oligonucleotide molecules in each of said plurality of said physically discrete wells.

20. The method of claim 19, wherein said sequencing said only one of said immobilized target oligonucleotide molecules comprises sequencing by synthesis.

21. The method of claim 16, further comprising sequencing said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells and creating multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells, wherein said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells are a single molecule species.

22. The method of claim 16, where the solid support is rigid or semi-rigid.

23. The method of claim 16, wherein

each of at least a portion of the plurality of physically discrete wells comprises a plurality of intermediate molecules selected from the group consisting of avidin, streptavidin, silane linkers, polyethylene glycol spacers, and polylysine, and

the immobilizing step comprises interacting the plurality of target oligonucleotide molecules with the plurality of intermediate molecules.

24. A method of producing a molecular array comprising:

providing a solid support comprising a plurality of physically discrete wells, wherein each of said plurality of physically discrete wells is arranged at predetermined locations of said solid support;

immobilizing a plurality of target oligonucleotide molecules into said physically discrete wells, thereby producing immobilized target oligonucleotide molecules and an array comprising said plurality of physically discrete wells having said immobilized target oligonucleotide molecules, wherein each of said plurality of physically discrete wells of the array contains only one of said immobilized target oligonucleotide molecules;

amplifying said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells and creating multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells, said multiple copies of said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells are a single molecule species; and

labeling at least a portion of said multiple copies of said only one of said immobilized target oligonucleotide molecules with one or more labels, thereby producing a molecular array comprising labeled said only one of said immobilized target oligonucleotide molecules in each of said plurality of physically discrete wells.