Patent ID: 8399188

Claim:
A method for sequencing a nucleic acid molecule, comprising: (a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, an extension enzyme, and a reversibly blocked nucleotide under conditions wherein said primer is extended to incorporate said nucleotide into a sequence complementary to said nucleic acid template, said reversibly blocked nucleotide comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker, and wherein, attached at the 2′ or 4′ position of said sugar moiety, is a reversible blocking group comprising —CH 2 —CH═CH 2 , —CH 2 —N 3 , —CH 2 —S—S-tBu, —CH 2 —S-Me, —CH 2 —O—CH 2 —CH 2 —CN, —CH 2 —CH 2 —CN, —C(O)—CH 3 , —CH 3 , 2-nitrobenzyl, tetrandyropyran, 4-nitrobenzoyl, 2-aminobenzoyl, —C(O)-linker-dye, 2-nitrobenzyl-linker-dye, —C(O)—O—CH 2 —CH═CH-linker-dye, —NH—C(O)—CH 3 , —F, —NH 2 , phosphate, or sulfate, (b) identifying the nucleotide in said sequence complementary to said nucleic acid template; (c1) enzymatically cleaving the peptide linker; and (c2) enzymatically removing the blocking group, wherein identifying said nucleotide identifies at least a portion of the sequence of said nucleic acid molecule.