De novo or “universal” sequencing array
First Claim
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1. 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 hybridizing region comprises 4-8 bases, which are bound to the spacer region and which can hybridize to a complementary region on the template nucleic acid molecule;
B) hybridizing said capture region of the template nucleic acid molecule to the capture moiety of the sequencing reagent;
C) hybridizing said sequence specific hybridizing region to said complementary region on the template nucleic acid molecule thereby forming a hybridized sequence specific region;
D) extending said hybridized sequence specific region by a polymerase with a chain terminating nucleotide thereby forming an extended sequence; and
E) determining the identity of said extended sequence.
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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.
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17 Claims
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1. A method of sequencing a template nucleic acid molecule on a combinatorial array comprising the steps:
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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 hybridizing region comprises 4-8 bases, which are bound to the spacer region and which can hybridize to a complementary region on the template nucleic acid molecule;
B) hybridizing said capture region of the template nucleic acid molecule to the capture moiety of the sequencing reagent;
C) hybridizing said sequence specific hybridizing region to said complementary region on the template nucleic acid molecule thereby forming a hybridized sequence specific region;
D) extending said hybridized sequence specific region by a polymerase with a chain terminating nucleotide thereby forming an extended sequence; and
E) determining the identity of said extended sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification