Sequencing of surface immobilized polymers utilizing microflourescence detection

Claim 1

A method of identifying nucleotide sequences contained within a plurality of different polynucleotides, said method comprising: (a) contacting said plurality of different polynucleotides with a surface of a solid substrate, said surface having attached thereto in spatially defined locations a plurality of different oligonucleotide primers, each of which has a free 3'-hydroxyl group and a defined nucleotide sequence and length, wherein said oligonucleotides are patterned on the surface of said substrate such that oligonucleotide primers with different nucleotide sequences are located in different locations; (b) hybridizing said oligonucleotide primers to regions of complementary nucleotide sequence contained in said plurality of different polynucleotides in a reaction mixture comprising a nucleic acid polymerase and a labeled nucleotide having a 3'-hydroxyl group reversibly blocked by a photoremovable protecting group, under conditions suitable for template directed polymerization to occur, such that said primers are elongated only if said labeled nucleotide is complementary to a nucleotide in said polynucelotide adjacent to a 3'-terminal nucleotide of said primers prior to elongation; (c) determining the location and thus the nucleotide sequence of the primer at that location at which said labeled nucleotide having a reversibly blocked 3'-hydroxyl group has been attached via a 5'-3' phosphodiester linkage to said 3'-hydroxyl group of said primer by detecting the presence of said label at said location, thereby determining a complementary sequence in said polynucleotide hybridized to said primer; (d) removing said photoremovable protecting group by irradiation; and (e) repeating steps (b) and (c) on the substrate resulting from step (d) using a labeled nucleotide having a reversibly blocked 3'-hydroxyl group, wherein said 3'-hydroxyl group is blocked by a photoremovable protecting group, and wherein said nucleotide is different from the nucleotide used in step (b).

Claim 2

A method of identifying nucleotide sequences contained within a plurality of different polynucleotides, said method comprising: (a) contacting said plurality of different polynucleotides with a surface of a solid substrate, said surface having attached thereto in spatially defined locations a plurality of different oligonucleotide primers, each of which has a free 3'-hydroxyl group and a defined nucleotide sequence and length, wherein said oligonucleotides are patterned on the surface of said substrate such that oligonucleotide primers with different nucleotide sequences are located in different locations; (b) hybridizing said oligonucleotide primers to regions of complementary nucleotide sequence contained in said plurality of different polynucleotides in a reaction mixture comprising a nucleic acid polymerase and a labeled nucleotide having a 3'-hydroxyl group reversibly blocked by a photoremovable protecting group, under conditions suitable for template directed polymerization to occur, such that said primers are elongated only if said labeled nucleotide is complementary to a nucleotide in said polynucelotide adjacent to a 3'-terminal nucleotide of said primers prior to elongation; (c) determining the location and thus the nucleotide sequence of the primer at that location at which said labeled nucleotide having a reversibly blocked 3'-hydroxyl group has been attached via a 5'-3' phosphodiester linkage to said 3'-hydroxyl group of said primer by detecting the presence of said label at said location, thereby determining a complementary sequence in said polynucleotide hybridized to said primer; (d) removing said photoremovable protecting group by irradiation; and (e) repeating steps (b) and (c) on the substrate resulting from step (d) using a labeled nucleotide having a reversibly blocked 3'-hydroxyl group, wherein said 3'-hydroxyl group is blocked by a photoremovable protecting group, and wherein said nucleotide is different from the nucleotide used in step (b).