Methods for making and imaging arrays that comprise a plurality of different biomolecules

• US 9,334,530 B2
• Filed: 05/13/2010
• Issued: 05/10/2016
• Est. Priority Date: 11/06/2007
• Status: Active Grant

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Lim, Mark J., Rothschild, Kenneth J.
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US12/779,493
• Publication Number:
  US 20100256015A1
• Time in Patent Office: 2,189 Days
• Field of Search: None
• US Class Current: 1/1
• 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) first and second proteins, ii) first and second photocleavable mass tags and iii) first and second beads, said beads comprising a binding agent used to bind said proteins to said beads, wherein said first protein is bound only to said first bead through said binding agent and said second protein is bound only to said second bead through said binding agent, wherein said first photocleavable mass tag is attached to said first bead and said second photocleavable mass tag is attached to said second bead and wherein said photocleavable mass tags attached to said first and second beads are of different mass, and wherein at least one of said first and second beads comprises a detectable label;

b) creating an array with said first and second beads;

c) imaging said array to determine the presence of said detectable label; and

d) subjecting said array directly to mass spectrometry analysis under conditions such that at least one of said first and second beads is identified by detection of its corresponding photocleavable mass tag, thereby identifying at least one of said first or second proteins in said imaged array.

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 selected from the group consisting of antibodies, avidin, NeutrAvidin and streptavidin.

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

5. The method of claim 1, wherein said detectable label is a fluorescent label.

6. The method of claim 1, wherein said photocleavable mass tags are photocleavable peptide mass tags.

7. The method of claim 1, wherein said proteins share a common epitope.

8. The method of claim 7, wherein said proteins are cell-free expressed proteins and said epitope is introduced during said cell-free expression.

20. The method of claim 1, wherein said imaging of step c) is fluorescent imaging.

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

a) providing i) first and second proteins, each protein comprising a shared epitope, ii) first and second photocleavable mass tags and iii) first and second beads, said beads comprising an antibody specific to said epitope, wherein said first bead is bound to said first protein though said antibody on said first bead and wherein said second bead is bound to said second protein through said antibody on said second bead, wherein said first photocleavable mass tag is attached to said first bead and said second photocleavable mass tag is attached to said second bead and wherein said photocleavable mass tags attached to said first and second beads are of different mass, and wherein at least one of said first and second beads comprises a detectable label;

b) creating an array with said first and second beads;

c) imaging said array to determine the presence of said detectable label; and

d) subjecting said array directly to mass spectrometry analysis under conditions such that at least one of said first and second beads is identified by detection of its corresponding photocleavable mass tag, thereby identifying at least one of said first or second proteins in said imaged array.

10. The method of claim 9, wherein said proteins are cell-free expressed proteins and said epitope is introduced during said cell-free expression.

11. The method of claim 9, wherein said shared epitope is the HSV epitope.

12. The method of claim 9, wherein said beads are agarose beads.

13. The method of claim 9, wherein said detectable label is a fluorescent label.

14. The method of claim 9, wherein said beads comprise avidin, NeutrAvidin or streptavidin.

15. The method of claim 14, wherein said antibody is biotin labeled and reacted with said avidin, NeutrAvidin or streptavidin beads in order to provide the beads of step a).

16. The method of claim 15, wherein said biotin is attached to said antibody via a photocleavable linker.

17. The method of claim 16, wherein said creating an array comprises cleaving said linker and phototransferring said antibody.

18. The method of claim 9, wherein said photocleavable mass tags are photocleavable peptide mass tags.

19. The method of claim 9, wherein said mass spectrometry analysis comprises MALDI mass spectrometry analysis.

21. The method of claim 9, wherein said imaging of step c) is fluorescent imaging.

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

a) providing i) first and second proteins, ii) first and second photocleavable mass tags, iii) a probe comprising a detectable label and iv) first and second beads, said beads comprising a binding agent used to bind said proteins to said beads, wherein said first protein is bound only to said first bead through said binding agent and said second protein is bound only to said second bead through said binding agent, wherein said first photocleavable mass tag is attached to said first bead and said second photocleavable mass tag is attached to said second bead and wherein said photocleavable mass tags attached to said first and second beads are of different mass;

b) creating an array with said first and second beads;

c) treating said array with said probe;

d) imaging said array to determine the presence of said detectable label; and

e) subjecting said array directly to mass spectrometry analysis under conditions such that at least one of said first and second beads is identified by detection of its corresponding photocleavable mass tag, thereby identifying at least one of said first or second proteins in said imaged array.