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

• US 20020042077A1
• Filed: 09/24/2001
• Published: 04/11/2002
• Est. Priority Date: 09/25/2000
• Status: Active Grant

• Current Assignee: Labcyte, Inc. (Danaher Corporation)
• Original Assignee: Picoliter Inc. (Danaher Corporation)
• Inventors: Ellson, Richard N.
• Granted Patent: US 6,806,051 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 a plurality of oligonucleotides attached to a substrate surface, wherein at least one of the oligonucleotides is a partially nonhybridizing oligonucleotide comprised of two discrete hybridizing segments with a nonhybridizing spacer segment therebetween.

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.

4. The oligonucleotide array of any one of claims 1, 2, or 3, wherein the nonhybridizing spacer segments are oligomeric segments comprised of a recurring single nucleotide.

5. The oligonucleotide array of claim 4, wherein the nonhybridizing 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 nonhybridizing 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 nonhybridizing 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 nonhybridizing 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 nonhybridizing 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.

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 partially nonhybridizing oligonucleotide.

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 partially nonhybridizing oligonucleotide are identical to each other.

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

21. The oligonucleotide array of claim 1, wherein the oligonucleotides 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 at least one partially nonhybridizing oligonucleotide includes at least one additional hybridizing segment, and wherein any two hybridizing segments are spaced apart by a nonhybridizing spacer segment.

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

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

38. 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.

28. An oligonucleotide array comprised of a plurality of partially nonhybridizing oligonucleotides attached to a substrate surface through a linking moiety, wherein each of the partially nonhybridizing oligonucleotides is comprised of alternating hybridizing segments and nonhybridizing spacer segments.

29. An oligonucleotide array comprised of a plurality of partially nonhybridizing oligonucleotides, each of which has the formula

SS-(L)_m-X¹-Y¹-X²-(Y²-X³)_n

wherein SS is a substrate surface, L is a linking moiety, X¹, X² and X³ are hybridizing oligonucleotide segments, Y¹ and Y² are nonhybridizing spacer segments, m is zero or 1, and n is an integer in the range of zero to approximately 50, and further wherein with any one partially nonhybridizing oligonucleotide, (a) the hybridizing segments may be the same or different, and (b) if more than one nonhybridizing segment is present, the nonhybridizing segments may be the same or different.

30. A multilayer oligonucleotide array comprised of a plurality of identical partially nonhybridizing oligonucleotides, each of which has the formula

SS-(L)_m-X¹-Y¹-X²-(Y²-X³)_n,

wherein SS is a substrate surface, L is a linking moiety, X¹, X² and X³ are hybridizing oligonucleotide segments, Y¹ and Y² are nonhybridizing spacer segments, m is zero or 1, and n is an integer in the range of zero to 50, wherein each nonhybridizing spacer segment Yⁱ of each oligonucleotide is covalently linked to the corresponding nonhybridizing spacer segment Yⁱ of at least one adjacent oligonucleotide through a linking moiety containing a cleavable site, and further wherein with any one partially nonhybridizing oligonucleotide, (a) the hybridizing segments may be the same or different, and (b) if more than one nonhybridizing segment is present, the nonhybridizing segments may be the same or different.

31. A substrate having an oligonucleotide attached to the surface thereof through a covalent or noncovalent bond, said oligonucleotide comprised of two discrete hybridizing segments with a nonhybridizing spacer segment therebetween.

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 a partially nonhybridizing oligonucleotide comprised of at least two discrete hybridizing segments with a nonhybridizing spacer segment therebetween, the method comprising:

(a) providing the plurality of oligonucleotides; and

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

33. The method of claim 32, wherein step (b) comprises applying each oligonucleotide to the substrate surface as a discrete fluid droplet.

34. The method of claim 33, 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.

36. The method of claim 35, wherein each nucleotidic monomer is applied to the substrate surface as a discrete fluid droplet.

37. The method of claim 36, wherein each nucleotidic monomer is applied to the substrate surface by applying focused acoustic energy to a fluid-containing reservoir containing a nucleotidic monomer and/or oligonucleotide segment to be applied to the substrate surface.

35. 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 a partially nonhybridizing oligonucleotide comprised of alternating hybridizing segments and nonhybridizing 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 nonhybridizing spacer segments.