Patent ID: 7622279

Claim:
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.