Method and apparatus for three label microarrays

• US 20050014147A1
• Filed: 08/16/2002
• Published: 01/20/2005
• Est. Priority Date: 08/21/2001
• Status: Abandoned Application

• Current Assignee: MCW Research Foundation Inc (Medical College of Wisconsin Incorporated)
• Original Assignee: MCW Research Foundation Inc (Medical College of Wisconsin Incorporated)
• Inventors: Hessner, Martin J
• Application Number:
  US10/486,244
• Publication Number:
  US 20050014147A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00693 Means for quality control
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

A method of and apparatus for directly visualizing printed microarrays are disclosed. In one embodiment, the method comprises the steps of (a) generating labeled probes labeled with a first label, (b) constructing a microarray with the labeled probes, wherein the microarray comprises a plurality of probe spots, and (c) examining the microarray to determine the amount of probe present at each probe spot.

Claims

1. A method of directly visualizing microarrays, comprising the steps of:

a) generating labeled probes labeled with a first label,

b) constructing a microarray with the labeled probes, wherein the microarray comprises a plurality of probe spots, and

c) examining the microarray to determine the amount of probe present at each probe spot.

2. The method of claim 1 wherein the probes are DNA molecules.

3. The method of claim 1 wherein the probes are selected from the group consisting of cDNA and oligonucleotides.

4. The method of claim 1 wherein the probe is selected from the group consisting of proteins and antibodies.

5. The method of claim 1 wherein the labeled probes are attached to the microarray surface via electrostatic and covalent bonds.

6. The method of claim 1 wherein the first label is fluorescent.

7. The method of claim 1 wherein the labeled probes are labeled with fluorescein.

8. The method of claim 1 wherein the label is selected from the group consisting of fluorescent, radioactive, phosphorescent and luminescent labels.

9. The method of claim 5 wherein the examination of step (c) is via the detection of relative fluorescence units and is by the use of a confocal laser scanner.

10. The method of claim 1 wherein a preferred amount of probe has been determined and the microarrays are evaluated using this preset amount.

11. The method of claim 5 wherein the fluorescently labeled probes of step (a) are generated via labeled primers.

12. The method of claim 2 wherein the labeled probes are between 10 and 100,000 base pairs in length.

13. The method of claim 2 wherein the probes comprise 1 label molecules per DNA strand on average.

14. The method of claim 1 additionally comprising the step of

(d) exposing the microarray to labeled target molecules wherein the labeled target molecules are labeled with a second and third label.

15. The method of claim 14 comprising the additional step of

(e) examining the microarray to determine the amount of target bound to the probes.

16. The method of claim 1 wherein the microarray comprises a poly-lysine-coated glass slide.

17. The method of claim 2 wherein DMSO/1.5 M betaine is used during the attachment of the probes to the microarray.

18. The method of claim 1 wherein step (c) comprises measurement of image quality as assessed by software which employs a spatial and intensity-dependent algorithm for spot detection and signal segmentation.

19. The method of claim 1 wherein the microarrays possess a density of 3,000-10,000 probes/slide.

20. A printed microarray comprising

a) a surface, and

b) labeled probes attached to the surface in a plurality of spots, wherein each probe is labeled with a first label, wherein the probe is selected from the group consisting of spotted oligonucleotides, cDNA, protein and antibodies.

21. The microarray of claim 20 wherein the probe is DNA.

22. The microarray of claim 20 wherein the probe is selected from the group consisting of nucleic acids, protein, and antibodies.

23. The array of claim 20 wherein the surface is a glass slide.

24. The array of claim 20 wherein the surface is coated with a coating selected from the group consisting of poly-L-lysine, aminosaline, epoxy, and aminoallyl.

25. The array of claim 20 wherein the first label is fluorescent.

26. The array of claim 25 wherein the first fluorescent label is fluorescein.

27. The array of claim 20 wherein the first label is selected from the group consisting of fluorescent, luminescent, radioactive or phosphorescent labels.