De novo or “universal” sequencing array

• US 6,322,968 B1
• Filed: 11/21/1997
• Issued: 11/27/2001
• Est. Priority Date: 11/21/1997
• 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, Head, Steven R., Karn, Jonathan, Goelet, Philip
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/976,427
• Time in Patent Office: 1,467 Days
• Field of Search: 536/24.3, 435/287.2, 435/6
• US Class Current: 435/6.12
• 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:

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, 4-8 bases in length, which is bound to the spacer region, and 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 reagent of claim 2, wherein said attachment moiety is at or near the 5′

terminus of the primer.

4. The sequencing reagent of claim 2, wherein said attachment moiety 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 capture moiety 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 nm 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.

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.

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. The 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.