De novo or universal sequencing array

• US 20020146704A1
• Filed: 06/29/2001
• Published: 10/10/2002
• Est. Priority Date: 11/21/1997
• Status: Active Grant

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Head, Steven R., Boyce-Jacino, Michael, Karn, Jonathan, Goelet, Philip
• Granted Patent: US 6,946,249 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

The present invention describes a novel nucleic acid sequencing reagent which consists of a capture moiety, a spacer region and a sequence specific hybridizing region of 4-8 bases. The nucleic acid sequencing reagent of the present invention can also contain an attachment moiety. The nucleic acid sequencing reagent can be arranged into a nested array. This array configuration can then be used to sequence a given template without prior knowledge (de novo) of the wild type or expected sequence in conjunction with primer extension in the presence of a labeled chain terminating nucleotide.

Claims

1. A sequencing reagent comprising one or more sequencing reagents wherein each reagent comprises:

i) a capture moiety which can form a stable complex with a region of a template nucleic acid molecule;

ii) a spacer region; and

iii) a sequence specific hybridizing region, wherein said sequence specific region comprises 4-8 bases which can hybridize to a complementary sequence on the template nucleic acid molecule.

2. The sequencing reagent of claim 1, wherein said reagent further comprises an attachment moiety which permits for attachment of the primer to a solid surface.

3. The sequencing regent of claim 2, wherein said modification is at or near the 5′

terminus of the primer.

4. The sequencing reagent of claim 2, wherein said modification is selected from the group consisting of amino, thiol, disulfide, avidin, strepavidin, iminobiotin, biotin, antibody and hapten.

5. The sequencing reagent of claim 1, wherein said capture moiety comprises either a nucleic acid sequence selected from the group of nucleotide analogs consisting of DNA, RNA and protein nucleic acid (PNA) or the group of non-nucleotide capture moieties consisting of biotin, iminobiotin, avidin, strepavidin, antibody, hapten, receptor, ligand and charged base and their analogs.

6. The sequencing reagent of claim 5, wherein said nucleic acid sequence comprises a sequence of 8-24 C bases.

7. The sequencing reagent of claim 1, wherein said capture moiety comprises a specific sequence complementary to a PCR primer, or portion thereof.

8. The sequencing reagent of claim 1, wherein said capture moiety is selected from the group consisting of biotin, iminobiotin, avidin, strepavidin, antibody, hapten, receptor, ligand and charged base.

9. The sequencing reagent of claim 1, wherein said spacer region is at least 10 Å

in length.

10. The sequencing reagent of claim 1, wherein said spacer region comprises a region of random, pseudo-random or non-random bases or analogs thereof.

11. The sequencing reagent of claim 1, wherein said spacer region comprises polyethylene glycol.

12. The sequencing reagent of claim 1, wherein said spacer region is designed to minimize template independent noise.

13. The sequencing reagent of claim 1, wherein said sequence specific region comprises 4-6 bases which can hybridize to a complementary sequence on the template nucleic acid molecule.

14. The sequencing reagent of claim 1, wherein said sequence specific region comprises 6 bases which can hybridize to a complementary sequence on the template nucleic acid molecule.

16. The array according to claim 15, wherein said array comprises a set, subset or combination of 4⁴-4⁸ different sequence reagents.

17. The array according to claim 15, wherein said array comprises 4⁴ different sequence reagents.

18. The array according to claim 15, wherein said array comprises 4⁵ different sequence reagents.

19. The array according to claim 15, wherein said array comprises 4⁶ different sequence reagents.

20. Tile array according to claim 15, wherein said array comprises 4⁷ different sequence reagents.

21. The array according to claim 15, wherein said array comprises 4⁸ different sequence reagents.

22. The array according to claim 15, wherein said array comprises an array of nested sequencing reagents.

24. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said sequencing reagent farther comprises an attachment moiety which permits for attachment of the primer to the surface.

25. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said capture sequence is on a first reagent and said sequence specific hybridizing region is on a second reagent.

26. The method of sequencing a template nucleic acid molecule according to claim 25, wherein said first reagent is co-attached to the solid phase and effectively brings the template into close proximity to the sequence specific hybridizing portion or the second primer.

27. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said modification of the primer is at or near the 5′

terminus of the sequencing reagent.

28. The method of sequencing a template nucleic acid molecule according to claim 27, wherein said modification is an amino, thiol, disulfide or biotin group.

29. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said capture moiety comprises a sequence of 8-24 C bases.

30. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said capture moiety comprises a specific sequence complementary to a PCR primer or portion thereof.

31. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said spacer region is at least 10Å

in length.

32. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said spacer region is a region of random, pseudo-random or non-random bases or analogs thereof.

33. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said spacer region is designed to minimize template independent noise.

34. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said solid surface is selected from the group consisting of glass and plastic.

35. The method of sequencing a template nucleic acid molecule according to claim 34, wherein said solid surface is fashioned as a 96-well or 384-well plate.

36. The method of sequencing a template nucleic acid molecule according to claim 34, wherein said plastic is polystyrene plastic.

37. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said solid surface is selected from the group consisting of a glass plate, a quartz wafer, a nylon membrane, a nitrocellulose membrane and a silicon wafer.

38. The method of sequencing a template nucleic acid molecule according to claim 23, wherein said chain terminating nucleotide is detectably labeled.

39. The method of sequencing a template nucleic acid molecule according to claim 38, wherein said detectably labeled chain terminating nucleotide is fluorescently labeled.

15. An array of sequencing array reagents, comprising an orderly arrangement of a plurality of sequencing array reagents immobilized to a solid surface wherein each of said sequencing array reagents comprises:

i) a capture moiety which can form a stable complex with a region of a template nucleic acid molecule;

ii) a spacer region; and

iii) a sequence specific hybridizing region, wherein said sequence specific region comprises 4-8 bases which can hybridize to a complementary sequence on the template nucleic acid molecule.

23. A method of sequencing a template nucleic acid molecule on a combinatorial array comprising the steps:

A) immobilizing a sequencing reagent to a solid surface, wherein said sequencing reagent comprises;

i) a capture moiety which can form a stable complex with a capture region of a template nucleic acid molecule;

ii) a spacer region; and

iii) a sequence specific hybridizing region, wherein said sequence specific region comprises 4-8 bases which can hybridize to a complementary region on the template nucleic acid molecule;

B) hybridizing said capture region of the template nucleic acid to the capture moiety of the sequencing reagent;

C) hybridizing said sequence specific hybridizing region to a complementary region on the template strand;

D) extending said hybridized sequence specific region by a polymerase with a chain terminating nucleotide;

E) determining the identity of said extended sequence.