Photocleavable Fluorescent Nucleotides for Dna Sequencing on Chip Constructed by Site-Specific Coupling Chemistry
First Claim
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1. A method for determining the sequence of a DNA, wherein (i) about 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 comprises a self-priming moiety, comprising performing the following steps for each nucleic acid residue of the DNA to be sequenced:
- (a) contacting the bound DNA with 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 being sequenced is bound to the DNA by the DNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;
(b) removing unbound nucleotide analogues; and
(c) determining the identity of the bound nucleotide analogue, thereby determining the sequence of the DNA.
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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.
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Citations
21 Claims
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1. A method for determining the sequence of a DNA, wherein (i) about 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 comprises a self-priming moiety, comprising performing the following steps for each nucleic acid residue of the DNA to be sequenced:
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(a) contacting the bound DNA with 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 being sequenced is bound to the DNA by the DNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;
(b) removing unbound nucleotide analogues; and
(c) determining the identity of the bound nucleotide analogue, thereby determining the sequence of the DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for determining the sequence of an RNA, wherein (i) about 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 comprises a self-priming moiety, comprising performing the following steps for each nucleic acid residue of the RNA to be sequenced:
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(a) contacting the bound RNA with 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 being sequenced is bound to the RNA by the RNA polymerase, and (ii) each of the four analogues has a pre-determined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;
(b) removing unbound nucleotide analogues; and
(c) determining the identity of the bound nucleotide analogue, thereby determining the sequence of the RNA. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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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.
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16. (canceled)
- 21. A compound comprising a modified nucleotide useful in sequencing a nucleic acid.
Specification