Patent ID: 9410889
Filing Date: 2016-08-09
CPC Classification: C12Q,G01N

Claim Text:
1. A method for sequencing a plurality of target nucleic acid molecules, comprising: delivering polymerase molecules to a plurality of spatially confined and separated source points provided within discrete cavities of a sample holder, each discrete cavity having a diameter smaller than a wavelength of an excitation light, wherein the polymerase molecules each have at least one photoactivatable linker and are secured to a nanobead by a linker, the nanobead sized to fit one bead in a cavity of the discrete cavities and deliver one polymerase molecule to the cavity; directing activation light to activate the photoactivatable linker and immobilize within the discrete cavities at least one polymerase molecule; subjecting the plurality of source points provided within the discrete cavities of the sample holder to nucleic acid polymerization reactions, wherein the source points each comprise fluorescence-labeled bases, primers, and at least a single target nucleic acid molecule, and wherein a majority of the plurality of source points each has a single polymerase molecule; directing the excitation light toward the sample holder at an angle off-axis with respect to a normal of the sample holder and achieving total internal reflection so that an amount of the excitation light is recycled within a substrate of the sample holder to illuminate the source points and to cause the source points to fluoresce while the discrete cavities reduce light scatter, wherein the directing excitation light further includes directing linearly polarized excitation light toward the sample holder; and collecting fluorescent signals from the illuminated source points and focusing the fluorescent signals onto at least one detector to form spatially resolved images of the source points on the at least one detector to determine time sequences of base incorporations in the polymerization reactions.