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US 2002/0164629 A1Published application
Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
AssigneeCalifornia Institute of Technology
InventorsStephen Quake, Ido Braslavsky, Benedict Hebert, Emil Kartalov
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Abstract
The invention provides methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension. Some applications of the invention utilize total internal reflection fluorescence microscopy to image polynucleotide molecules at single molecule resolution.
Claims28
A method of analyzing sequence of a target polynucleotide, comprising:
- (a) providing a primed target polynucleotide immobilized to a surface of a substrate;
- wherein the target polynucleotide is attached to the surface with single molecule resolution;
- (b) adding a first fluorescently labeled nucleotide to the surface of the substrate under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;
- (c) determining presence or absence of a fluorescence signal on the surface where the target polynucleotide is immobilized, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide;
- and
- (d) repeating steps (b)-(c) with a further fluorescently labeled nucleotide, the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer.
3 more elements
A method of analyzing sequence of a target polynucleotide, comprising:
- (a) providing a primed target polynucleotide immobilized to a surface of a substrate;
- wherein the target polynucleotide is attached to the surface with single molecule resolution;
- (b) adding four types of nucleotides to the surface of the substrate under conditions whereby nucleotides attach to the primer dynamically, when complementary nucleotides are present in the target polynucleotide;
- and
- (c) monitoring in a time course of incorporation of fluorescent signals into the immobilized primer.
2 more elements
An apparatus for analyzing the sequence of a target polynucleotide, comprising:
- (a) a flow cell comprising a substrate for immobilizing the target polynucleotide with single molecule resolution;
- (b) an inlet port and an outlet port, said inlet port and outlet port being in fluid communication with said flow cell for flowing fluids into and through said flow cell;
- (c) a light source for illuminating the surface of the substrate;
- and
- (d) a detection system for detecting a signal from said surface.
2 more elements
Dependencies are inferred from the claim text.