Polynucleotide arrays and methods of making and using the same

• US 6,245,518 B1
• Filed: 12/06/1999
• Issued: 06/12/2001
• Est. Priority Date: 12/11/1998
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Baier, Joerg
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): KIM, YOUNG J
• Application Number:
  US09/454,822
• Time in Patent Office: 554 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.33
• US Class Current: 435/6.11
• 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 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00515 in the shape of strings
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• 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 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

The present invention relates to methods of using labeled tracers to generate spatially addressable arrays of immobilized molecules, particularly polynucleotides, that can be normalized for differences in immobilization efficiencies at different addresses in the array. It also relates to the arrays generated by the method and to use of these arrays to enhance discrimination in array-based assays, particularly the discrimination between perfectly matched hybrids and hybrids containing a single mismatch in nucleic acid hybridization assays.

Claims

1. A spatially-addressable array of compounds, comprising a substrate having directly attached thereon, optionally by a linker at each of a plurality of distinct addresses a compound and a tracer moiety, wherein the structures of the compounds are identifiable by their spatial addresses, and wherein the amount of the tracer moiety attached at each address is proportional to the amount of compounds attached at that address.

2. The array of claim 1 which is a one-dimensional array.

3. The array of claim 2 in which the substrate is a solid-phase synthesis support, a glass fiber or a capillary tube.

4. The array of claim 1 which is a two-dimensional array.

5. The array of claim 1 in which the substrate is a glass or plastic sheet.

6. The array of claim 1, in which the directly attached compounds are polynucleotides.

7. The array of claim 6 in which the polynucleotides are covalently attached to the substrate, optionally by way of a linker.

8. The array of claim 7, in which the polynucleotides are covalently attached to the substrate via their 5′

or 3′

terminal nucleotide.

9. The array of claim 7 which comprises 10 to 10⁶ different polynucleotides.

10. The array of claim 7 in which each immobilized polynucleotide is independently 6 to 20 nucleotides in length.

11. The array of claim 10, in which all of the directly attached polynucleotides are the same length.

12. The array of claim 10, which comprises a complete set of polynucleotides 6-10 nucleotides in length.

13. The array of claim 6 in which the polynucleotide directly attached, optionally by a linker, at one or more address is a mixture of polynucleotides.

14. The array of claim 13, in which the polynucleotide directly attached, optionally by a linker, at each address is a mixture of polynucleotides.

15. The array of claim 13 or 14, in which the mixture is of the formula:

N_xB_yN_z, wherein each N represents any of the five encoding bases and varies for the polynucleotides in a given mixtures, B represents any of the five encoding bases and is the same for each of the polynucleotides in a given mixture, and x, y, and z are each independently integers.

16. The array of claim 1 in which the tracer moiety comprises a fluorophore.

17. The array of claim 6 in which the tracer moiety is a fluorescently-labeled polynucleotide.

18. The array of claim 17 in which the fluorescently-labeled polynucleotide directly attached, optionally by a linker, at each address has the same nucleotide sequence as the polynucleotide directly attached, optionally by a linker at that address.

19. The array of claim 17 in which the fluorescent label is TAMRA.

20. The array of claim 17, in which the same fluorescently-labeled polynucleotide is directly attached, optionally by a linker, at each address.

21. The array of claim 20, in which the fluorescently-labeled polynucleotide comprises a mixture of fluorescently-labeled polynucleotides.

22. The array of claim 21, in which the mixture of fluorescently labeled polynucleotides comprises:

NNNGGCAT-F,

NNNCGGAG-F,

NNNAACTG-F,

NNNATGAA-F,

NNNTGTAC-F,

NNNACTGG-F,

NNNGAACC-F,

NNNTACAG-F,

NNNCTGGA-F, and

NNNCCGGA-F,

wherein each N represents any of the five encoding bases and F is a fluorophore.

23. The array of claim 22, in which F is TAMRA.

27. A method of increasing the accuracy of an array-based assay comprising:

contacting an array according to claim 1 with a an analyte compound that is capable of generating an assay signal upon interacting with a compound of the array; and

normalizing the assay signals to account for differences in the amounts of compounds immobilized at different addresses in the array, thereby increasing the accuracy of the assay.

28. The method of claim 27 in which the assay signals are normalized by obtaining the ratio of the assay signal intensity at an address to the background signal intensity at that address.

29. The method of claim 28 in which the background signal intensity at an address is obtained by measuring the signal intensity of that address prior to contacting the array with the analyte compound.

30. A method of normalizing hybridization signals in an array-based hybridization experiment, comprising the steps of:

contacting an array of immobilized polynucleotides according to claim 6 with a target nucleic acid under conditions in which addresses of the array bearing immobilized polynucleotides that are complimentary to a region of the target nucleic acid produce a detectable hybridization signal; and

normalizing the hybridization signal at an address by obtaining the ratio of the hybridization signal intensity at the address to the background signal intensity at that address.

31. The method of claim 30 in which the background signal intensity of the address is obtained by measuring the signal intensity at the address prior to contacting the array with the target nucleic acid.

32. The method of claim 30 in which the target nucleic acid is labeled.

33. The method of claim 30 in which the array is further contacted with a set of labeled solution-phase polynucleotide probes and labeled probes and array polynucleotides that hybridize adjacently to the same target nucleic acid molecule are covalently joined.

24. A method of making a spatially-addressable array of compounds, comprising the steps of:

(i) directly attaching, optionally by a linker, at a first address of a substrate a first compound and a first tracer moiety; and

(ii) directly attaching, optionally by a linker, a second address of a substrate a second compound and a second tracer moiety.

25. In a method of making spatially addressable array of polynucleotides by directly attaching, optionally by a linker pre-synthesized polynucleotides at a discrete spatial address on a substrate, the improvement comprising directly attaching, optionally by a linker an amount of a tracer moiety at each spatial address that is proportional to the amount of polynucleotide attached at that address.

26. In a method of making a spatially addressable array of compounds by in situ synthesis, the improvement comprising directly attaching optionally by a linker at each spatial address of the array an amount of a tracer moiety that is proportional to the amount of a product of the in situ synthesis directly attached, optionally by a linker, at that address.