Arrays and methods of use

• US 9,556,429 B2
• Filed: 02/06/2015
• Issued: 01/31/2017
• 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:
  US14/616,224
• Publication Number:
  US 20150203903A1
• Time in Patent Office: 725 Days
• Field of Search: 435/6.1, 435/6.11, 435/91.1, 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, 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 performing an assay on a molecular array comprising:

(a) producing a molecular array comprising producing labeled, immobilised oligonucleotides on a solid phase at least by

(i) immobilising a plurality of oligonucleotides to the solid phase, thereby forming two or more separate and discrete elements on the solid phase, and

(ii) labeling the plurality of oligonucleotides with one or more labels before or after the immobilising step, thereby producing the labeled, immobilised oligonucleotides in said two or more separate and discrete elements,

wherein at least a portion of the labeled, immobilised oligonucleotides within each of the elements are individually optically resolvable from another portion of the labeled, immobilised oligonucleotides within each of the elements, and each of the elements comprises a plurality of the labeled, immobilised oligonucleotides;

(b) analysing whether at least a portion of the labeled, immobilised oligonucleotides on the solid phase are individually optically resolvable from another portion of the labeled, immobilised oligonucleotides on the solid phase, whereby said at least a portion of the labeled, immobilised oligonucleotides on each of at least two elements of the elements is individually optically resolvable from said another portion of the labeled, immobilised oligonucleotides; and

(c) performing the assay comprising counting the number or measuring a total intensity of at least a portion of the labeled, immobilised oligonucleotides within each of said at least two elements of the elements.

2. The method according to claim 1, wherein

said at least two elements of the elements are spatially addressable, and

sequence identity of said at least a portion of the labeled, immobilised oligonucleotides in each of the at least two elements of the elements is specified by a location of each of said at least two elements of the elements.

3. The method according to claim 2, wherein

said one or more labels comprise different labels and the labeled, immobilised oligonucleotides comprise one or more first labeled, immobilised oligonucleotide and one of more second labeled, immobilised oligonucleotide which have the different labels, and

each of said at least two elements of the elements comprises the one or more first labeled, immobilised oligonucleotide and the one or more second labeled, immobilised oligonucleotide.

4. The method according to claim 3, wherein the method further comprises comparing a counted number of the one or more first labeled, immobilised oligonucleotide to a counted number of the one or more second labeled, immobilised oligonucleotide in at least one of said at least two elements of the elements.

5. The method according to claim 2, wherein the labeled, immobilised oligonucleotides in said at least two elements of the elements are not sequenced.

6. The method according to claim 1, wherein the molecular array can be used for detecting a polymorphism of the labeled, immobilised oligonucleotides.

7. The method according to claim 1, further comprising determining a frequency of an allele of the labeled, immobilised oligonucleotides.

8. The method according to claim 1, wherein each of the one or more labels comprises a fluorescent molecule, nanoparticle, or nanorod.

9. The method according to claim 1, wherein the labeling step is performed by hybridizing a labeled probe to the at least a portion of the plurality of oligonucleotides.

10. The method according to claim 1, wherein the counting step comprises single molecule counting the at least a portion of the labeled, immobilised oligonucleotides within each of said at least two elements of the elements which is individually optically resolvable from another portion of the labeled, immobilised oligonucleotides.

11. The method according to claim 1, wherein the counting step comprises counting a number of signals from the one or more labels of the at least a portion of the labeled, immobilised oligonucleotides within each of said at least two elements of the elements which is individually optically resolvable from another portion of the labeled, immobilised oligonucleotides.

12. The method according to claim 1, wherein the two or more separate and discrete elements are separated by a raised region or an etched trench.

13. The method according to claim 1, wherein at least a portion of the labeled, immobilized oligonucleotides are ligated to target nucleic acids, thereby forming probe-target molecule complexes.

14. The method according to claim 13, wherein the probe-target molecule complexes comprise circularized DNA.

15. The method according to claim 14 further comprising amplifying at least a portion of the probe-target molecule complexes by rolling circle amplification.

16. The method according to claim 13, wherein the producing step further comprises primer extension of at least a portion of the probe-target molecule complexes with labeled primers.

17. The method according to claim 1, wherein the oligonucleotides comprises a biotin or a SH group or an amine group or a phenylboronic acid (PBA) or an acrydite and the at least a portion of the oligonucleotides are immobilised to the solid support by interacting the biotin with Avidin, Strepatavidin or Neutravidin on the solid support;

by covalently linking the SH group via a disulphide bond to a SH-surface on the solid support;

covalently linking the amine group to an activated carboxylate or an aldehyde group on the solid support;

by complexing the PBA with salicylhydroxamic acid (SHA) on the solid support; and

by reacting the acrydite with thiol or silane surface on the solid support or co-polymerizing with acrylamide monomer on the solid support to form polyacrylamide.

18. A method of performing an assay on a molecular array comprising:

(a) producing a molecular array comprising producing labeled, immobilised oligonucleotides on a solid phase at least by

(i) immobilising a plurality of linearized oligonucleotides to the solid phase, thereby forming two or more separate and discrete elements on the solid phase and

(ii) labeling the linearized oligonucleotides with one or more labels before or after the immobilising step, thereby producing the labeled, immobilised oligonucleotides in said two or more separate and discrete elements,

wherein at least a portion of the labeled, immobilised oligonucleotides on the solid phase are individually optically resolvable from another portion of the labeled, immobilised oligonucleotides on the solid phase, and each of the elements comprises a plurality of the labeled, immobilised oligonucleotides;

(b) analysing whether at least a portion of the labeled, immobilised oligonucleotides on the solid phase are individually optically resolvable from another portion of the labeled, immobilised oligonucleotides on the solid phase, whereby said at least a portion of the labeled, immobilised oligonucleotides on each of at least two elements of the elements is individually optically resolvable from said another portion of the labeled, immobilised oligonucleotides; and

(c) performing the assay comprising counting the number or measuring a total intensity of at least a portion of the labeled, immobilised oligonucleotides.

19. The method according to claim 18, wherein said one or more labels comprise different labels, and the labeled, immobilised oligonucleotides comprise one or more first labeled, immobilised oligonucleotide and one of more second labeled, immobilised oligonucleotide which have the different labels.

20. The method according to claim 19, wherein the method further comprises comparing a counted number of the one or more first labeled, immobilised oligonucleotide to a counted number of the one or more second labeled, immobilised oligonucleotide.

21. The method according to claim 18, further comprising determining a frequency of an allele of the labeled, immobilised oligonucleotides.

22. The method according to claim 18, wherein each of the one or more labels comprises a fluorescent molecule, nanoparticle, or nanorod.

23. The method according to claim 18, wherein the labeled, immobilised oligonucleotides in said at least two elements of the elements are not sequenced.

24. The method according to claim 18, wherein the labeling step is performed by hybridizing a labeled probe to the at least a portion of the plurality of linearized oligonucleotides.

25. The method according to claim 18, wherein the counting step comprises single molecule counting the at least a portion of the labeled, immobilised oligonucleotides within each of said at least two elements of the elements which is individually optically resolvable from another portion of the labeled, immobilised oligonucleotides.

26. The method according to claim 18, wherein the counting step comprises counting a number of signals from the one or more labels of the at least a portion of the labeled, immobilised oligonucleotides within each of said at least two elements of the elements which is individually optically resolvable from another portion of the labeled, immobilised oligonucleotides.

27. The method according to claim 18, wherein the two or more separate and discrete elements are separated by a raised region or an etched trench.