Method of sequencing a nucleic acid
Assignee454 Life Sciences Corporation
InventorsJonathan M. Rothberg, Joel S. Bader, Scott B. Dewell, Keith McDade, John W. Simpson, Jan Berka, Christopher M. Colangelo
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Abstract
Disclosed herein are methods and apparatuses for sequencing a nucleic acid. These methods permit a very large number of independent sequencing reactions to be arrayed in parallel, permitting simultaneous sequencing of a very large number (>10,000) of different oligonucleotides.
Claims87
A method for sequencing a nucleic acid, the method comprising:
- providing one or more nucleic acid anchor primers;
- providing a plurality of single-stranded circular nucleic acid templates;
- annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;
- combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;
- annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;
- extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetennined nucleotide triphosphate is incorporated onto the 3′ end of said sequencing primer, a sequencing reaction byproduct;
- and
- identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.
A method for analyzing a mixture of polymers, the method comprising:
- providing a plurality of at least 1,000 independent reaction sites attached to a solid substrate, wherein said independent reaction sites comprise a reaction center and said independent reactions sites are separated by distances that eliminate diffusion of reactants and products between sites;
- contacting said plurality of reaction sites with a fluid comprising said mixture of polymers;
- and
- separating said polymers from the fluid and from each other by attachment to the reaction sites, thereby analyzing said mixture of biopolymers.
A substrate for analyzing a nucleic acid, the substrate comprising:
- a cavitated fiber Optic surface;
- and
- a nucleic acid sequence linked to the fiber optic surface
A substrate with a cavitated surface comprising 103 or more groups of oligonucleotides attached to the surface in discrete known regions, the 103 or more groups of oligonucleotides occupying a total area of less than 1 cm2 on said substrate, said groups of oligonucleotides having different nucleotide sequences.
An array of more than 1,000 different groups of oligonucleotide molecules with known sequences covalently coupled to a surface of a cavitated substrate, said groups of oligonucleotide molecules each in discrete known regions and differing from other groups of oligonucleotide molecules in monomer sequence, each of said discrete known regions being an area of less than about 0.01 cm2 and each discrete known region comprising oligonucleotides of known sequence, said different groups occupying a total area of less than 1 cm2.
An apparatus for analyzing a nuclcic acid sequence, the apparatus comprising:
- a reagent delivery chamber, wherein the chamber includes a substrate with immobilized nucleic acids;
- a conduit in communication with the reagent delivery chamber;
- an imaging system in communication with the reagent delivery chamber;
- and
- a data collection system in communication with the imaging system.
An apparatus for processing a plurality of analytes, the apparatus comprising:
- a flow chamber having disposed therein a substrate comprising a plurality of cavitated surfaces, said cavitated surfaces having disposed thereon nucleic acid molecules;
- fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes anchored to the plurality of microparticles are exposed to the reagents;
- and
- detection means for detecting a sequence of optical signals from each microparticle of the plurality, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte anchored thereto, wherein said detection means is in communication with the cavitated surfaces.
The apparatus of claim 85, wherein said detection means further comprises signal tracking means for correlating said optical signals from each of said micropaiticles in each of said digital images to form for each said microparticle of said plurality a sequence of said optical signals.
The apparatus of claim 87, wherein said signal tracking means is a CCD camera.
Dependencies are inferred from the claim text.