Covalent attachment of unmodified nucleic acids to silanized solid phase surfaces

• US 6,387,626 B1
• Filed: 08/14/2000
• Issued: 05/14/2002
• Est. Priority Date: 06/06/1997
• Status: Expired due to Fees

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Orchid Biosciences Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Boyce-Jacino, Michael T., Shi, Jufang
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/638,436
• Time in Patent Office: 638 Days
• Field of Search: 435/6, 435/91.2, 536/25.4, 436/94
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to a simple, cost effective method for immobilizing synthetic nucleic acid molecules onto a solid support The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.

Claims

1. A method for immobilizing an unmodified nucleic acid molecule to a solid phase, which comprises the steps of:

(A) coating said solid phase with a silane and allowing said silane-coated solid phase to cure; and

(B) coupling unmodified nucleic acid molecules having either a terminal 3′

OH or a terminal 5′

OH to said silane-coated solid phase.

2. The method of claim 1, wherein in step A, said coating occurs in the presence of an acidic buffer of aqueous ethanol for at least about 30 minutes.

3. The method of claim 1, wherein in step A, said silane is selected from the group consisting of mercapto-alkyl-trimethoxysilane and glycidoxy-alkyl-silane.

4. The method of claim 1, wherein in step A, said silane is 3-mercapto-propyl-trimethoxysilane.

5. The method of claim 1, wherein in step A, said silane is 3-glycidoxy propyl-silane.

6. The method of claim 4, wherein in step A, said curing occurs for about 24 hours in the presence of a dry inert gas consisting of Ar or N₂.

7. The method of claim 5, wherein in step A, said curing occurs at a temperature of from about 60°

C. to about 70°

C. for about 10-14 hours.

8. The method of claim 1, wherein in step B, said coupling occurs in alkaline solution.

9. The method of claim 8, wherein in step B, said alkaline solution comprises from about 0.0001 M to about 5 M NaOH.

10. The method of claim 8, wherein in step B, said alkaline solution comprises NaOH, KOH, and LiOH.

11. The method of claim 1, wherein in step B, said nucleic acid molecules comprise oligonucleotides.

12. The method of claim 11, wherein said oligonucleotides have a concentration of from about 1.0 to about 10 uM.

13. The method of claim 1, wherein in step B, said unmodified nucleic acid molecules are coupled to said silane-coated solid phase by a covalent linkage selected from the group consisting of ether and thioether linkages.

14. The method of claim 4, wherein in step B, said unmodified nucleic acid molecules are coupled to said solid phase by a covalent thioether linkage.

15. The method of claim 5, wherein in step B, said unmodified nucleic acid molecules are coupled to said solid phase by a covalent ether linkage.

16. The method of claim 1, wherein said solid phase is glass.

17. The method of claim 16, wherein said glass is a microscope slide.

18. The method of claim 1, wherein said solid-phase is plastic.

19. The method of claim 18, wherein said plastic is polystyrene plastic.

20. The method of claim 1, wherein said solid-phase is selected from the group consisting of a bead, a plate, a column, a pin and a dipstick.

21. The method of claim 1, wherein step B said coupling is employed to make an array of immobilized nucleic acid molecules.

22. The method of claim 1, wherein said unmodified nucleic acid molecules coupled to said silane-coated solid phase are immobilized polynucleotides and wherein said method further comprises the steps of:

(C) capturing from a solution at least one strand of a specific polynucleotide analyte by hybridization to said immobilized polynucleotide; and

(D) detecting the presence of the captured analyte.

23. The method of claim 22, wherein step C further comprises the steps of:

(C′

(1)) amplifying a specific region of a specific genome using a polymerase chain reaction, said region having a sequence complementary to said immobilized polynucleotide; and

(C′

(2)) capturing from solution at least one strand of said amplification product by hybridization to said immobilized polynucleotide; and

wherein step D further comprises the step of

(D′

) detecting the presence of the captured amplification product.

24. The method of claim 23, wherein said method further comprises the steps of:

(E) incubating a sample of nucleic acid of a target organism, containing a single nucleotide polymorphism in the presence of said immobilized polynucleotide and at least one dideoxynucleotide derivative, under conditions sufficient to permit a polymerase mediated, template-dependent extension of said immobilized polynucleotide, said extension causing the incorporation of a single dideoxynucleotide to the 3′

-terminus of said immobilized polynucleotide, said single dideoxynucleotide being complementary to the single nucleotide of the polymorphic site of said polymorphism;

(F) permitting said template-dependent extension of said immobilized polynucleotide molecule, and said incorporation of said single dideoxynucleotide; and

(G) determining the identity of the nucleotide incorporated into said polymorphic site, said identified nucleotide being complementary to said nucleotide of said polymorphic site.

25. The method of claim 19, wherein said polystyrene support is a microwell plate.

26. The method of claim 19, wherein said polystyrene support is an array designed to fit into a microwell plate.

27. The method of claim 1, wherein said coating step is by means of an aerosol, a vaporization means or any other mechanical means.