Methods For Making And Imaging Arrays

• US 20100256015A1
• Filed: 05/13/2010
• Published: 10/07/2010
• Est. Priority Date: 11/06/2007
• Status: Active Grant

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Lim, Mark J., Rothschild, Kenneth J.
• Granted Patent: US 9,334,530 B2
• Time in Patent Office: Days
• US Class Current: 506/12
• 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 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Methods of amplifying nucleic acid on a solid support are described. Beads and template, each in known concentrations, are employed so a range of template to bead ratios can be exploited. Where the beads contain primers, the template can be amplified. After amplification, non-covalently bound template is removed, so as to leave beads with extended primers (or beads with primers that were not extended).

Claims

1. A method of making and imaging arrays, comprising:

a) providing i) a plurality of different biomolecules, and ii) a plurality of beads, said beads comprising a binding agent capable of binding to said biomolecules;

b) capturing a first biomolecule on a first bead with said binding agent so as to create a first reacted bead;

c) capturing a second biomolecule on a second bead with said binding agent so as to create a second reacted bead;

d) creating an array with said first and second reacted beads;

e) imaging said array; and

f) subjecting said array to mass spec analysis.

2. The method of claim 1, wherein said binding agent is an amine-reactive NHS ester.

3. The method of claim 1, wherein said binding agent is an antibody.

4. The method of claim 1, wherein said beads are agarose beads.

5. The method of claim 1, wherein said beads are fluorescent.

6. The method of claim 1, wherein said array of step d) comprises a plurality of codes of unique and distinguishable masses.

7. The method of claim 6, wherein said codes are photocleavable peptide codes.

8. The method of claim 7, wherein said peptide codes are released and detected during the mass spec analysis of step f).

9. The method of claim 1, wherein said plurality of different biomolecules comprise a plurality of different proteins.

10. The method of claim 9, wherein said proteins share a common epitope.

11. The method of claim 10, wherein said proteins are cell-free expressed proteins and said epitope is introduced during said cell-free expression.

12. A method of making and imaging arrays, comprising:

a) providing i) a plurality of different proteins, each protein comprising a shared epitope, and ii) a plurality of beads, said beads comprising an antibody specific to said epitope;

b) capturing a first protein on a first bead with said antibody so as to create a first reacted bead;

c) capturing a second protein on a second bead with said antibody so as to create a second reacted bead;

d) creating an array with said first and second reacted beads;

e) imaging said array; and

f) subjecting said array to mass spec analysis.

13. The method of claim 12, wherein said proteins are cell-free expressed proteins and said epitope is introduced during said cell-free expression.

14. The method of claim 12, wherein said shared epitope is the HSV epitope.

15. The method of claim 12, wherein said beads are agarose beads.

16. The method of claim 12, wherein said beads are fluorescent.

17. The method of claim 12, wherein said beads comprise avidin.

18. The method of claim 17, wherein said antibody is biotin labeled and reacted with said avidin beads in order to provide the beads of step a).

19. The method of claim 18, wherein said biotin is attached to said antibody via a photocleavable linker.

20. The method of claim 19, wherein said creating an array comprises cleaving said linker and phototransferring said antibody.

21. The method of claim 12, wherein said array of step d) comprises a plurality of peptide codes of unique and distinguishable masses.

22. The method of claim 21, wherein said peptide codes are photocleavably attached to said array.

23. The method of claim 22, wherein said peptide codes are released and detected during the mass spec analysis of step f).

24. The method of claim 12, wherein said mass spec analysis comprises MALDI mass spec analysis.

25. An array comprising first and second beads, said first bead comprising a first mass tag and a first biomolecule, said second bead comprising a second mass tag and a second biomolecule, wherein said first and second biomolecules are different and said first and second mass tags are of different mass.

26. The array of claim 26, wherein said first and second mass tags are peptide mass tags.

27. A method of making an array, comprising a) providing a mixture comprising first and second beads, said first bead comprising a first mass tag and a first biomolecule, said second bead comprising a second mass tag and a second biomolecule, wherein said first and second biomolecules and said first and second mass tags are different;

b) making an array with said mixture.

28. The method of claim 27, wherein said first and second mass tags are peptide mass tags.

29. A method of analyzing an array, comprising a) providing a mixture comprising first and second beads, said first bead comprising a first mass tag and a first biomolecule, said second bead comprising a second mass tag and a second biomolecule, wherein said first and second biomolecules and said first and second mass tags are different;

b) making an array with said mixture; and

c) subjecting said array to mass spec analysis.

30. The method of claim 29, wherein said first and second mass tags are peptide mass tags.

31. The method of claim 29, wherein said first and second mass tags are photocleavable.

32. The method of claim 29, wherein said first and second biomolecules are captured proteins.

33. The method of claim 29, wherein said biomolecules are attached via a photocleavable linker.

34. The method of claim 33, wherein said linker is cleaved and the biomolecule is released directly from the bead for mass spec analysis.

35. The method of claim 29, wherein said mass spec analysis comprises MALDI mass spec analysis.