Arrays of partially nonhybridizing oligonucleotides and preparation thereof using focused acoustic energy

• US 6,806,051 B2
• Filed: 09/24/2001
• Issued: 10/19/2004
• Est. Priority Date: 09/25/2000
• Status: Expired due to Term

• Current Assignee: Labcyte, Inc. (Danaher Corporation)
• Original Assignee: Picoliter Inc. (Danaher Corporation)
• Inventors: Ellson, Richard N.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/962,731
• Publication Number:
  US 20020042077A1
• Time in Patent Office: 1,121 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/6, 536/21, 536/911, 536/91.2, 536/287.2
• US Class Current: 435/6.18
• 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 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/0036 Nozzles
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• 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 2219/00608 DNA chips
• 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 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

Partially nonhybridizing oligonucleotides are provided that contain two or more hybridizing segments, with any two hybridizing segments separated by a nonhybridizing spacer segment, i.e., a nucleotidic or nonnucleotidic segment that has little or no likelihood of binding to an oligonucleotide sequence found in nature. Oligonucleotide arrays are also provided in which at least one of the oligonucleotides of the array is a partially nonhybridizing oligonucleotide. The partially nonhybridizing oligonucleotides serve as multifunctional probes wherein each hybridizing segment of a single partially nonhybridizing oligonucleotide serves as an individual probe. Also provided are methods for preparing and using the partially nonhybridizing oligonucleotides and arrays formed therewith. A particularly preferred method of array fabrication involves the use of focused acoustic energy.

Claims

1. An oligonucleotide array comprised of each attached to a substrate surface through the 3′

-hydroxyl group or the 5′

-hydroxyl group of the oligonucleotide, wherein at least one of the oligonucleotides is a multifunctional oligonucleotide probe comprised of at least two discrete hybridizing segments with a nonhybridizing spacer segment present between any two hybridizing segments, wherein (a) at least two of the hybridizing segments within the same multifunctional oligonucleotide probe are identical, or (b) at least two of the hybridizing segments within the same multifunctional oligonucleotide probe are different and capable of hybridizing to different nucleic acid sequences, such that each hybridizing segment within the same multifunctional probe is capable of use as an individual probe, and further wherein the nonhybridizing spacer segments are selected from nonnucleotidic spacer segments and nucleotidic spacer segments having an equilibrium constant of less than or equal to 1% of the binding constant of the hybridizing segments to complementary oligonucleotide segments in an oligonucleotide analyte.

2. The oligonucleotide array of claim 1, wherein the hybridizing segments are oligonucleotide segments in the range of approximately 8 to approximately 400 nucleotides in length.

3. The oligonucleotide array of claim 2, wherein the hybridizing segments are oligonucleotide segments in the range of approximately 16 to approximately 80 nucleotides in length.

15. The oligonucleotide array of claim 1, wherein each oligonucleotide of the array is attached to the substrate surface by a covalent bond.

16. The oligonucleotide array of claim 1, wherein each oligonucleotide of the array is attached to the substrate surface by a non-covalent bond.

17. The oligonucleotide array of claim 1, wherein each oligonucleotide of the array is comprised of a multifunctional oligonucleotide probe.

18. The oligonucleotide array of claim 1, wherein each oligonucleotide of the array is different.

19. The oligonucleotide array of claim 18, wherein the hybridizing segments within any one multifunctional oligonucleotide probe are identical to each other.

20. The oligonucleotide array of claim 1, wherein the hybridizing segments within any one multifunctional oligonucleotide probe are different from each other.

21. The oligonucleotide array of claim 1, wherein the oligonucleotides of the array are comprised of a plurality of oligonucleotide groups, wherein (a) all oligonucleotides within any one group are different, and (b) each oligonucleotide group is identical to each other oligonucleotide group.

22. The oligonucleotide array of claim 1, wherein the oligonucleotides are present at a density in the range of approximately 10 to approximately 250,000 oligonucleotides per square centimeter of substrate surface.

23. The oligonucleotide array of claim 22, wherein the oligonucleotides are present at a density of at least about 1,000,000 oligonucleotides per square centimeter of substrate surface.

24. The oligonucleotide array of claim 23, wherein the oligonucleotides are present at a density of at least about 1,500,000 oligonucleotides per square centimeter of substrate surface.

25. The oligonucleotide array of claim 1, wherein the substrate surface is comprised of a porous material.

26. The oligonucleotide array of claim 25, wherein the porous material is a permeable material.

35. In a method for conducting a solid phase hybridization assay by contacting a sample with an oligonucleotide array under hybridizing conditions and thereafter detecting any hybridization events, the improvement which comprises employing the oligonucleotide array of claim 1 as the oligonucleotide array.

4. An oligonucleotide array comprised of a plurality of oligonucleotides attached to a substrate surface, wherein at least one of the oligonucleotides is comprised of at least two discrete hybridizing segments with a spacer segment present between any two hybridizing segments, wherein the spacer segments are oligomeric segments consisting of approximately 9 to approximately 50 identical nucleotides.

5. The oligonucleotide array of claim 4, wherein the spacer segments are comprised of(A)_n wherein n is in the range of approximately 9 to approximately 50.

6. The oligonucleotide array of claim 5, wherein n is in the range of approximately 9 to approximately 20.

7. The oligonucleotide array of claim 4, wherein the spacer segments are comprised of (T)_n wherein n is in the range of approximately 9 to approximately 50.

8. The oligonucleotide array of claim 7, wherein n is in the range of approximately 9 to approximately 20.

9. The oligonucleotide array of claim 4, wherein the spacer segments are comprised of (G)_n wherein n is in the range of approximately 9 to approximately 50.

10. The oligonucleotide array of claim 9, wherein n is in the range of approximately 9 to approximately 20.

11. The oligonucleotide array of claim 4, wherein the spacer segments are comprised of (C)_n wherein n is in the range of approximately 9 to approximately 50.

12. The oligonucleotide array of claim 11, wherein n is in the range of approximately 9 to approximately 20.

13. The oligonucleotide array of claim 4, wherein the spacer segments are comprised of (N)_n wherein n is in the range of approximately 9 to approximately 50 and N is a nonnaturally occurring nucleotide or a modified naturally occurring nucleotide.

14. The oligonucleotide array of claim 13, wherein n is in the range of approximately 9 to approximately 20.

27. An oligonucleotide array comprised of a plurality of oligonucleotides, each of which has the formula

28. A multilayer oligonucleotide array comprised of a plurality of oligonucleotides, each of which has the formula

29. A method for synthesizing an oligonucleotide array comprised of a plurality of oligonucleotides attached to a substrate surface wherein at least one of the oligonucleotides is comprised of at least two discrete hybridizing segments with a spacer segment therebetween, wherein the spacer segment is an oligomeric segment consisting of approximately 9 to approximately 50 identical nucleotides, the method comprising:

(a) providing the plurality of oligonucleotides; and

(b) attaching each of the oligonucleotides to a particular location on the substrate surface.

30. The method of claim 29, wherein step (b) comprises applying each oligonucleotide to the substrate surface as a discrete fluid droplet.

31. The method of claim 30, wherein step (b) is carried out by applying focused acoustic energy to each of a plurality of fluid-containing reservoirs, each of said reservoirs containing an oligonucleotide to be applied to the substrate surface.

32. A method for synthesizing an oligonucleotide array comprised of a plurality of oligonucleotides attached to a substrate surface wherein at least one of the oligonucleotides is comprised of alternating hybridizing segments and spacer segments, wherein each spacer segment is an oligomeric segment consisting of approximately 9 to approximately 50 identical nucleotides, the method comprising successively coupling individual nucleotidic monomers and/or oligonucleotide segments to a plurality of support-bound initial nucleotidic monomers, wherein the nucleotidic monomers and/or oligonucleotide segments are selected so as to provide at least one oligonucleotide with alternating hybridizing segments and spacer segments.

33. The method of claim 32, wherein each nucleotidic monomer and/or oligonucleotide segment is applied to the substrate surface as a discrete fluid droplet.

34. The method of claim 33, wherein focused acoustic energy is applied to a fluid-containing reservoir containing the nucleotidic monomer and/or oligonucleotide segment to generate the discrete fluid droplet.

36. A method for synthesizing an oligonucleotide array comprised of a plurality of oligonucleotides attached to a substrate surface wherein at least one of the oligonucleotides is comprised of at least two discrete hybridizing segments with a spacer segment therebetween, the method comprising:

(a) providing a plurality of fluid-containing reservoirs each containing one of the plurality of oligonucleotides;

(b) applying focused acoustic energy to each of the reservoirs to eject a discrete fluid droplet from each reservoir toward a particular location on the substrate surface for deposition thereon, wherein each discrete fluid droplet contains an oligonucleotide, and further wherein the spacer segment is selected from nonnucleotidic spacer segments and nucleotidic spacer segments having an equilibrium binding constant of less than or equal to 1% of the binding constant of the hybridizing segments to complementary oligonucleotide segments in an oligonucleotide analyte.

37. A method for synthesizing an oligonucleotide array comprised of a plurality of oligonucleotides attached to a substrate surface wherein at least one of the oligonucleotides is comprised of alternating hybridizing segments and spacer segments, the method comprising successively coupling individual nucleotidic monomers and/or oligonucleotide segments to a plurality of support-bound initial nucleotidic monomers, wherein the nucleotidic monomers and/or oligonucleotide segments are selected so as to provide at least one oligonucleotide with alternating hybridizing segments and spacer segments, wherein each individual monomer and/or oligonucleotide segment is applied to the substrate as a discrete fluid droplet generated by applying focused acoustic energy to each of a plurality of fluid-containing reservoirs each containing one of said monomers and/or oligonucleotide segments, and further wherein the spacer segments are selected from nonnucleotidic spacer segments or nucleotidic spacer segments having an equilibrium binding constant of less than or equal to 1% of the binding constant of the hybridizing segments to complementary oligonucleotide segments in an oligonucleotide analyte.

38. An oligonucleotide array comprised of a plurality of multifunctional oligonucleotide probes each covalently attached through an optional linking moiety to a substrate surface through the 3′

-hydroxyl group or the 5′

-hydroxyl group of the probe, wherein each probe is comprised of at least two discrete hybridizing segments with a nucleotidic spacer segment present between any two hybridizing segments, wherein all hybridizing segments within the same probe are different and capable of use as individual probes and further wherein the nucleotidic spacer segment has an equilibrium binding constant of less than or equal to 1% of the binding constant of the hybridizing segments to complementary oligonucleotide segments in an oligonucleotide analyte.