Methods for testing oligonucleotide arrays

• US 6,238,862 B1
• Filed: 12/19/1997
• Issued: 05/29/2001
• Est. Priority Date: 09/18/1995
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Barone, Anthony D., Lockhart, David J., McGall, Glenn, Chee, Mark, Diggelmann, Martin, Caviani Pease, Ann Maria
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/995,265
• Time in Patent Office: 1,257 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/25.3, 536/25.32, 436/518, 436/528, 436/527
• US Class Current: 435/6.14
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00626 Covalent
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00693 Means for quality control
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y10S 435/807 Gas detection apparatus

Abstract

Methods for testing oligonucleotide arrays are disclosed including methods for testing the efficiency of nucleotide coupling; methods for testing amounts of deprotected oligonucleotides; methods for determining amounts of depurinated oligonucleotides; and methods of detecting the presence of cleavable structural features, such as double-stranded nucleic acids.

Claims

1. A quality control process for manufacturing nucleic acid probe arrays comprising manufacturing nucleic acid probe arrays by spatially directed nucleic acid synthesis in high volume and testing arrays selected from among the high volume manufactured.

2. The method of claim 1 wherein the spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

3. The method of claim 1 wherein selected arrays are tested for the efficiency of monomer coupling during spatially directed nucleic acid synthesis.

4. The method of claim 1 wherein selected arrays are tested for the amount of deprotection of nucleic acids in the selected arrays.

5. The method of claim 1 wherein selected arrays are tested for the amount of double stranded nucleic acids in the nucleic acid probe array.

37. The method of claim 1 wherein high volume is the manufacture of at least 10 nucleic acid probe arrays per day from a single fabrication machine or in a single fabrication facility.

38. The method of claim 1 wherein high volume is the manufacture of at least 50 nucleic acid probe arrays per day from a single fabrication machine or in a single fabrication facility.

39. The method of claim 1 wherein high volume is the manufacture of at least 500 nucleic acid probe arrays per day from a single fabrication machine or in a single fabrication facility.

6. A testing method comprising:

providing a substrate having a surface with linkers having active sites for nucleic acid synthesis;

synthesizing an ensemble of sequence-specific nucleic acids on the substrate by spatially directed nucleic acid synthesis;

exposing a first area of the substrate to a first test condition;

exposing a second area of the substrate to a second test condition; and

determining the relative amount of nucleic acids having a structural feature in the first and second area, wherein the structural feature is not monomer coupling and the test condition is not exposure to a nucleic acid probe having a sequence complementary to a sequence in the array;

whereby the relative amount indicates the relative efficiency to cause the appearance of the structural feature.

7. The method of claim 6 wherein spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

8. The method of claim 6 comprising synthesizing at least two ensembles of different sequence-specific nucleic acids on the substrate.

9. The method of claim 6 wherein the nucleic acids have active sites for attaching a detectable label and the step of determining the amount of nucleic acids having the structural feature comprises exposing the nucleic acids to a detectable label under conditions for attachment of the label to nucleic acids having the structural feature, and determining the amount of detectable label attached.

10. The method of claim 9 wherein the detectable label is a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.

11. The method of claim 10 wherein the label is a fluorescein, a rhodamine, a polymethine dye derivative or a phosphor.

12. A method for testing the efficiency of monomer coupling in the synthesis of a nucleic acid probe array by spatially directed nucleic acid synthesis comprising the steps of:

providing a substrate having a surface having linkers with active sites;

coupling first protected monomers to active sites in a first area and at least one second area of the substrate and capping unreacted, unprotected active sites;

deprotecting active sites in the second area(s), coupling second protected monomers to active sites in the second area(s) and capping unreacted, unprotected active sites in the second area(s);

optionally repeating the previous step in at least one subsequent area of the substrate and capping unreacted, unprotected active sites in the subsequent area(s);

determining the amount of competent, uncapped active sites at at least two areas; and

comparing the amounts determined, wherein the comparative amount indicates the efficiency of monomer coupling between the two areas.

13. The method of claim 12 wherein spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

14. The method of claim 13 wherein the step of determining the amounts of uncapped active sites comprises the steps of deprotecting active sites in the two areas, exposing deprotected active sites in the two areas to a detectable label for attachment, and determining the amount of detectable label attached in the areas.

15. The method of claim 14 wherein the detectable label is a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.

16. The method of claim 15 wherein the label is a fluorescein, a rhodamine, a polymethine dye derivative or a phosphor.

17. A method for testing the efficiency of monomer coupling in the synthesis of a nucleic acid probe array by spatially directed nucleic acid synthesis comprising the steps of:

providing a substrate having a surface having cleavable linkers wherein the linkers comprise detectable labels releasable upon cleavage of the linkers and active sites for monomer coupling;

coupling at least one monomer to the active sites whereby the detectable labels are coupled to the monomer and capping unreacted, unprotected active sites after at least one coupling step;

cleaving the cleavable linkers to release detectably labelled nucleic acids;

determining the lengths of the released nucleic acids; and

comparing the amounts of nucleic acids having a first length and a second length, wherein the comparative amount indicates the efficiency of monomer coupling between the nucleic acids of the first length and the second length.

18. The method of claim 17 wherein spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

19. The method of claim 18 wherein the cleavable linker is a sulfone, a disulfide or an ester.

20. The method of claim 18 wherein the lengths are determined by determining the mass of the released nucleic acids by HPLC.

21. The method of claim 18 wherein the detectable label is a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.

22. The method of claim 21 wherein the label is a fluorescein, a rhodamine, a polymethine dye derivative or a phosphor.

23. A method for comparing the relative efficiency of two test conditions to cause deprotection of nucleic acids synthesized on a substrate by spatially directed nucleic acid synthesis comprising the steps of:

providing a substrate on which an ensemble of sequence-specific nucleic acids has been synthesized, wherein the active sites on the free terminal nucleotides of the nucleic acids bear a protecting group;

exposing a first area of the substrate to a first test condition;

exposing a second area of the substrate to a second test condition; and

determining the amount of unprotected active sites in the first and second areas, whereby the relative amount indicates the relative efficiency.

24. The method of claim 23 wherein spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

25. The method of claim 24 wherein the step of determining the amount of unprotected active sites comprises the step of exposing the nucleic acids in the area to a detectable label for attachment to unprotected active sites, and determining the amount of detectable label attached in the area.

26. The method of claim 25 wherein the detectable label is a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.

27. The method of claim 26 wherein the label is a fluorescein, a rhodamine, a polymethine dye derivative or a phosphor.

28. The method of claim 23 wherein a substrate is provided on which at least two ensembles of different sequence-specific nucleic acids have been synthesized by spatially directed nucleic acid synthesis, and at least two areas of the substrate, each area having a different sequence-specific nucleic acid, are exposed to the same test condition.

29. The method of claim 24 wherein the test condition is exposure to light, acid, base, nucleophilic agent, reducing agent or oxidizing agent.

30. A method of determining whether an ensemble of nucleic acids synthesized on a substrate by spatially directed nucleic acid synthesis contains double-stranded nucleic acids formed from the nucleic acids within themselves or between nucleic acids in the ensemble comprising:

providing a substrate on which an ensemble of sequence-specific nucleic acids has been synthesized in an area of the substrate, wherein nucleic acids in the ensemble are attached to a detectable label that is released from the area upon cleavage of the nucleic acids;

contacting the ensemble with an agent that cleaves double-stranded nucleic acids, whereby cleavage of nucleic acids formed into double stranded nucleic acids releases detectable label from the area; and

determining the amount of detectable label remaining the area, whereby the amount of detectable label is inversely related to the amount of double-stranded nucleic acids.

31. The method of claim 30 wherein spatially directed nucleic acid synthesis is performed by light-directed nucleic acid synthesis.

32. The method of claim 31 wherein the detectable label is a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.

33. The method of claim 32 wherein the label is a fluorescein, a rhodamine, a polymethine dye derivative or a phosphor.

34. The method of claim 31 comprising synthesizing at least two ensembles of different a sequence-specific nucleic acids on the substrate.

35. The method of claim 31 wherein the cleavage agent is an endonuclease.

36. The method of claim 35 wherein the endonuclease is a restriction endonuclease.