De novo or “universal” sequencing array
View Patent ↗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.
1. A sequencing reagent-template complex comprising:
i) a template nucleic acid molecule; and
ii) a sequencing reagent, the sequencing reagent comprising:
a. a capture moiety that can form a stable complex with a region of said template nucleic acid molecule;
b. a spacer region, said spacer region comprising nucleotide bases; and
c. a sequence specific hybridizing region, 4-8 nucleotide bases in length that is bound to the spacer region, and that is capable of hybridizing to a complementary sequence on the template nucleic acid molecule;
wherein said spacer region is not complementary to a region on the template nucleic acid molecule immediately adjacent to the sequence of the template nucleic acid molecule that is capable of hybridizing to said sequence specific hybridizing region.
2. The sequencing reagent-template complex of claim 1 , wherein said reagent is further comprised of an attachment moiety.
3. The sequencing reagent-template complex of claim 2 , wherein said attachment moiety is bound to said capture moiety.
4. The sequencing reagent-template complex 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-template complex of claim 1 , wherein said capture moiety comprises a nucleic acid sequence selected from the group of nucleotide analogs consisting of DNA, RNA and protein nucleic acid (PNA).
6. The sequencing reagent-template complex of claim 1 , wherein said capture moiety comprises a non-nucleotide capture moiety selected from the group consisting of biotin, iminobiotin, avidin, strepavidin, antibody, hapten, receptor, ligand and charged base and their analogs.
7. The sequencing reagent-template complex of claim 1 , wherein said capture moiety comprises a nucleic acid sequence of 8-24 bases.
8. The sequencing reagent-template complex of claim 1 , wherein said capture moiety is bound to said spacer region.
9. The sequencing reagent-template complex of claim 1 , wherein said spacer region is at least 10 nm in length.
10. The sequencing reagent-template complex of claim 1 , wherein said spacer consists essentially of 7 or 8 nucleotide bases.
11. A sequencing reagent-template complex comprising:
i) a template nucleic acid molecule, and
ii) a sequencing reagent, the sequencing reagent comprising:
a. a capture moiety that can form a stable complex with a region of a template nucleic acid molecule;
b. a spacer region, said spacer region comprising non-nucleotide moieties; and
c. a sequence specific hybridizing region, 4-8 nucleotide bases in length that is bound to the spacer region, and that is capable of hybridizing to a complementary sequence on the template nucleic acid molecule.
12. The sequencing reagent-template complex of claim 11 , wherein said reagent is further comprised of an attachment moiety.
13. The sequencing reagent-template complex of claim 12 , wherein said attachment moiety is bound to said capture moiety.
14. The sequencing reagent-template complex of claim 12 , wherein said attachment moiety is selected from the group consisting of amino, thiol, disulfide, avidin, strepavidin, iminobiotin, biotin, antibody and hapten.
15. The sequencing reagent-template complex of claim 11 , 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).
16. The sequencing reagent-template complex of claim 11 , wherein said capture moiety comprises a non-nucleotide capture moiety selected from the group consisting of biotin, iminobiotin, avidin, strepavidin, antibody, hapten, receptor, ligand and charged base and their analogs.
17. The sequencing reagent-template complex of claim 11 , wherein said capture moiety comprises a nucleic acid sequence of 8-24 bases.
18. The sequencing reagent-template complex of claim 11 , wherein said capture moiety is bound to said spacer region.