Patent ID: 7270951
Filing Date: 2007-09-18
Classification: B82Y,C12Q,G01N

Abstract:
1. A method for nucleotide base sequencing comprising the sequential steps of: (a) immobilizing a plurality of polymerases on a solid support in the absence of nucleic acid wherein each polymerase is immobilized in a reaction center of said solid support, and wherein said solid support comprises a plurality of reaction centers each containing a single polymerase located at an optically resolvable distance from each other; (b) providing a single nucleic acid sample for each of the plurality of said polymerases and a plurality of different oligonucleotide primers, wherein each of the nucleic acid sample hybridizes to a single oligonucleotide primer; (c) providing four different nucleotides, each nucleotide being differentially-labeled with a detachable labeling group and blocked at the 3′ portion with a detachable blocking group, wherein the polymerase extends the primer hybridized to the nucleic acid sample with a single differentially-labeled nucleotide that is complementary to the sample nucleic acid thereby creating a single detachable labeling group attached to the solid support; (d) removing nucleotides that have not been incorporated in the primer; (e) detecting the single labeled nucleotide incorporated into the elongating primer in each of reaction centers by detecting the single labeling group attached to the solid support, thereby identifying the complement of the labeled 3′-blocked nucleotide at each said reaction center; (f) separating the 3′ blocking group and the labeling group from the incorporated nucleotide; (g) removing the separated 3′ blocking group and the separated labeling group of step (f) to produce an unlabeled nucleic acid sample; (h) confirming separation and removal of the 3′ blocking group from the nucleotide incorporated in the primer of each reaction center by detecting for the presence of the single labeled nucleotide in each of the reaction centers wherein the presence of a labeled nucleotide indicates that the step of separating the labeling group from the incorporated nucleotide was not successful; and (i) repeating steps (c) through (h) until either no new nucleotides are incorporated in step (c) or the 3′ blocking group persists in not being separated and removed in steps (f) and (g), whereby the order in which the labeled nucleotides in step (e) are detected in a reaction center corresponds to the complement of the sequence of at least a portion of the nucleic acid sample in that reaction center.