Sequencing of surface immobilized polymers utilizing microflourescence detection
First Claim
Patent Images
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).
5 Assignments
0 Petitions
Accused Products
Abstract
Means for simultaneous parallel sequence analysis of a large number of biological polymer macromolecules. Apparatus and methods may use fluorescent labels in repetitive chemistry to determine terminal monomers on solid phase immobilized polymers. Reagents which specifically recognize terminal monomers are used to label polymers at defined positions on a solid substrate.
-
Citations
7 Claims
-
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). - View Dependent Claims (2, 3, 4, 5, 6, 7)
-
Specification