Photocleavable fluorescent nucleotides for DNA sequencing on chip constructed by site-specific coupling chemistry
First Claim
Patent Images
1. A method for determining the sequence of a DNA, wherein (i) 1000 or fewer copies of the DNA are bound to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii) each copy of the DNA is a denatured single-stranded template and comprises a 5′
- -phosphorylated self-priming moiety covalently linked to a 3′
-end of the DNA, comprising performing the following steps;
(a) contacting the bound DNA with a DNA polymerase and four photocleavable fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (i) the nucleotide analogues consist of an analogue of G, an analogue of C, an analogue of T and an analogue of A, so that a nucleotide analogue complementary to the residue of the single-stranded template being sequenced is incorporated into the DNA extension product on the 5′
-phosphorylated self-priming moiety of the bound DNA by the DNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence emission wavelength which is different than the fluorescence emission wavelengths of the other three analogues;
(b) removing the unincorporated nucleotide analogues not incorporated into the DNA extension product on the 5′
-phosphorylated self-priming moiety of the bound DNA;
(c) determining the identity of the incorporated nucleotide analogue and;
(d) repeating steps (a) to (c) for incorporation of the next nucleotide analogue complementary to the subsequent base of the bound single stranded template DNA,thereby determining the sequence of the DNA.
3 Assignments
0 Petitions
Accused Products
Abstract
This invention provides a method for determining the sequence of a DNA or an RNA, wherein (i) about 1000 or fewer copies of the DNA or RNA are bound to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii) each copy of the DNA or RNA comprises a self-priming moiety.
141 Citations
21 Claims
-
1. A method for determining the sequence of a DNA, wherein (i) 1000 or fewer copies of the DNA are bound to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii) each copy of the DNA is a denatured single-stranded template and comprises a 5′
- -phosphorylated self-priming moiety covalently linked to a 3′
-end of the DNA, comprising performing the following steps;(a) contacting the bound DNA with a DNA polymerase and four photocleavable fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (i) the nucleotide analogues consist of an analogue of G, an analogue of C, an analogue of T and an analogue of A, so that a nucleotide analogue complementary to the residue of the single-stranded template being sequenced is incorporated into the DNA extension product on the 5′
-phosphorylated self-priming moiety of the bound DNA by the DNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence emission wavelength which is different than the fluorescence emission wavelengths of the other three analogues;(b) removing the unincorporated nucleotide analogues not incorporated into the DNA extension product on the 5′
-phosphorylated self-priming moiety of the bound DNA;(c) determining the identity of the incorporated nucleotide analogue and; (d) repeating steps (a) to (c) for incorporation of the next nucleotide analogue complementary to the subsequent base of the bound single stranded template DNA, thereby determining the sequence of the DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- -phosphorylated self-priming moiety covalently linked to a 3′
-
8. A method for determining the sequence of an RNA, wherein (i) 1000 or fewer copies of the RNA are bound to a solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry and (ii) each copy of the RNA is a single-stranded template and comprises a 5′
- -phosphorylated self-priming moiety covalently linked to a 3′
-end of the bound RNA, comprising performing the following steps;(a) contacting the bound RNA with a RNA polymerase and four photocleavable fluorescent nucleotide analogues under conditions permitting the RNA polymerase to catalyze RNA synthesis, wherein (i) the nucleotide analogues consist of an analogue of G, an analogue of C, an analogue of U and an analogue of A, so that a nucleotide analogue complementary to the residue of the single-stranded template being sequenced is incorporated into the RNA extension product on the 5′
-phosphorylated self-priming moiety of the bound RNA by the RNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence emission wavelength which is different than the fluorescence emission wavelengths of the other three analogues;(b) removing the unincorporated nucleotide analogues not incorporated into the RNA extension product on the 5′
phosphorylated self-priming moiety of the bound RNA;(c) determining the identity of the incorporated nucleotide analogue and; (d) repeating steps (a) to (c) for incorporation of the next nucleotide analogue complementary to the subsequent base of the bound single stranded template RNA, thereby determining the sequence of the RNA. - View Dependent Claims (9, 10, 11, 12, 13, 14)
- -phosphorylated self-priming moiety covalently linked to a 3′
-
15. A composition of matter comprising a solid substrate having a DNA or an RNA bound thereto via 1,3-dipolar azide-alkyne cycloaddition chemistry, wherein (i) about 1000 or fewer copies of the DNA or the RNA are bound to the solid substrate, and (ii) each copy of the DNA or the RNA comprises a self-priming moiety.
- 16. A compound having the structure:
-
18. A composition of matter comprising a solid substrate having a RNA bound thereto via 1,3-dipolar azide-alkyne cycloaddition chemistry, wherein (i) about 1000 or fewer copies of the RNA are bound to the solid substrate, and (ii) each copy of the RNA comprises a self-priming moiety.
Specification