Method of sequencing a nucleic acid

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. The method includes annealing a population of circular nucleic acid molecules to a plurality of anchor primers linked to a solid support, and amplifying those members of the population of circular nucleic acid molecules which anneal to the target nucleic acid, and then sequencing the amplified molecules by detecting the presence of a sequence byproduct.

What is claimed is:  
     
         1 . A method for sequencing a nucleic acid, the method comprising: 
 providing one or more or more nucleic acid anchor primers linked to a solid support;    providing a plurality of 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 generate multiple copies of the circular nucleic acid template;    annealing an effective amount of a sequencing primer to the circular nucleic acid template to yield a primed sequencing primer-circular nucleic acid template complex;    extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and a sequencing reaction byproduct; and    identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.    
     
     
         2 . The method of  claim 1 , wherein the circular nucleic acid template is single-stranded DNA.  
     
     
         3 . The method of  claim 1 , wherein the circular nucleic acid template is an open circle nucleic acid.  
     
     
         4 . The method of  claim 1 , wherein the circular nucleic acid template is a closed circle nucleic acid.  
     
     
         5 . The method of  claim 1 , wherein the circular nucleic acid template is genomic DNA.  
     
     
         6 . The method of  claim 1 , wherein the circular nucleic acid template is cDNA.  
     
     
         7 . The method of  claim 1 , wherein the circular nucleic acid is 10-200 nucleotides in length.  
     
     
         8 . The method of  claim 1 , wherein the circular nucleic acid is 10-100 nucleotides in length.  
     
     
         9 . The method of  claim 1 , wherein the circular nucleic acid is 10-50 nucleotides in length.  
     
     
         10 . The method of  claim 1 , wherein the multiple copies are generated by a polymerase chain reaction.  
     
     
         11 . The method of  claim 1 , wherein the primed circular template is extended by rolling circle amplification to yield a single-stranded concatamer of the annealed circular nucleic acid template.  
     
     
         12 . The method of  claim 11 , further comprising: 
 annealing a reverse primer to the single-stranded concatamer to yield a primed concatamer template, and    combining the primed concatamer template with a polymerase enzyme to generate multiple copies of the concatamer template.    
     
     
         13 . The method of  claim 1 , wherein the sequencing byproduct is pyrophosphate.  
     
     
         14 . The method of  claim 13 , wherein the pyrophosphate is detected by contacting the sequencing byproduct with ATP sulfurylase under conditions sufficient to form ATP.  
     
     
         15 . The method of  claim 14 , wherein the ATP is detected with luciferase.  
     
     
         16 . The method of  claim 13 , further comprising apyrase.  
     
     
         17 . The method of  claim 13 , further comprising washing the sequencing product with a wash buffer.  
     
     
         18 . The method of  claim 17 , wherein the wash buffer includes apyrase.  
     
     
         19 . The method of  claim 1 , wherein the anchor primer sequence includes a biotin group.  
     
     
         20 . The method of  claim 19 , wherein the biotin group on the anchor primer is linked to an avidin group on the solid support.  
     
     
         21 . The method of  claim 1 , wherein the anchor primer is conjugated to a biotin-BSA moiety.  
     
     
         22 . The method of  claim 21 , wherein the biotin-BSA moiety on the anchor primer is linked to an avidin-biotin group on the solid support.  
     
     
         23 . The method of  claim 21 , wherein the biotin-BSA moiety on the anchor primer is linked to a BSA group on the solid support in the presence of silane.  
     
     
         24 . The method of  claim 1 , wherein the solid support includes at least one optical fiber.  
     
     
         25 . The method of  claim 1 , wherein the sequencing primer is extended in the presence of a dATP analog.  
     
     
         26 . The method of  claim 25 , wherein the dATP analog is adenosine 5′-phosphosulfate (APS).  
     
     
         27 . The method of  claim 1 , wherein the solid substrate includes two or more anchoring primers separated by approximately 10 μm to approximately 200 μm.  
     
     
         28 . The method of  claim 27 , wherein the solid substrate includes two or more anchoring primers separated by approximately 50 μm to approximately 150 μm.  
     
     
         29 . The method of  claim 27 , wherein the solid substrate includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.  
     
     
         30 . The method of  claim 1 , wherein the solid support matrix comprises of a plurality of anchor pads that are covalently linked to the solid support.  
     
     
         31 . The method of  claim 30 , wherein the surface area of each anchor pad is approximately 10 μm 2 .  
     
     
         32 . The method of  claim 30 , wherein and each pad is separated from one another by a distance ranging from approximately 50 μm to approximately 150 μm.  
     
     
         33 . 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.    
     
     
         34 . The substrate of  claim 33 , wherein the substrate comprises a plurality of fiber optic surfaces.  
     
     
         35 . The substrate of  claim 33 , wherein the nucleic acid sequence is an anchor primer.  
     
     
         36 . The substrate of  claim 33 , wherein the fiber optic surface includes two or more anchoring primers separated by approximately 10 μm to approximately 200 μm.  
     
     
         37 . The substrate of  claim 33 , wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.  
     
     
         38 . The substrate of  claim 33 , wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μm.  
     
     
         39 . The substrate of  claim 33 , wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μm to approximately 150 μm.  
     
     
         40 . The substrate of  claim 39 , wherein the surface area of each pad is approximately 10 μm 2 .  
     
     
         41 . A substrate with a cavitated surface comprising 10 3  or more groups of oligonucleotides covalently attached to the surface in discrete known regions, the 10 3  or more groups of oligonucleotides occupying a total area of less than 1 cm 2  on said substrate, said groups of oligonucleotides having different nucleotide sequences.  
     
     
         42 . The substrate as recited in  claim 41  wherein said substrate comprises 10 4  or more different groups of sequences in discrete known regions.  
     
     
         43 . The substrate as recited in  claim 1  wherein said substrate comprises 10 5  or more different groups of oligonucleotides with known sequences in discrete known regions.  
     
     
         44 . The substrate as recited in  claim 1  wherein the groups of oligonucleotides are attached to the surface by a linker.  
     
     
         45 . 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 cm 2  and each discrete known region comprising oligonucleotides of known sequence, said different groups occupying a total area of less than 1 cm 2 .  
     
     
         46 . The array as recited in  claim 45  wherein said area is less than 10,000 microns 2 .  
     
     
         47 . The array as recited in  claim 46  made by the process of: 
 exposing a first region of said substrate to light to remove photoremovable group from nucleic acids in said first region, and not exposing a second region of said surface to light;  
 covalently coupling a first nucleotide to said nucleic acids on said part of said substrate exposed to light, said first nucleotide covalently coupled to said photoremovable group;  
 exposing a part of said first region of said substrate to light, and not exposing another part of said first region of said substrate to light to remove said photoremovable groups; covalently coupling a second nucleotide to said part of said first region exposed to light; and  
 repeating said steps of exposing said substrate to light and covalently coupling nucleotides until said more than 500 different groups of nucleotides are formed on said surface.  
 
     
     
         48 . The array as recited in  claim 46  comprising more than 10,000 groups of oligonucleotides of known sequences.  
     
     
         49 . An apparatus for analyzing a nucleic acid sequence, the apparatus comprising: 
 a perfusion chamber, wherein the chamber includes a nucleic acid substrate;    a conduit in communication with the perfusion chamber;    an imaging system in communication with the perfusion chamber; and    a data collection system in communication with the imaging system.    
     
     
         50 . The apparatus of  claim 49 , wherein the substrate is a planar substrate.  
     
     
         51 . The apparatus of  claim 49 , wherein the imaging system is a fiber optic system.  
     
     
         52 . The apparatus of  claim 49 , wherein the substrate comprises 
 a cavitated fiber optic surface in communication with said imaging system; and    a nucleic acid sequence linked to the fiber optic surface.    
     
     
         53 . The apparatus of  claim 49 , wherein the substrate comprises a plurality of fiber optic surfaces, said fiber optic surfaces being in communication with said imaging system.  
     
     
         54 . The apparatus of  claim 49 , wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μm to approximately 150 μm.  
     
     
         55 . The apparatus of  claim 49 , wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μm.  
     
     
         56 . The apparatus of  claim 49 , wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μm to approximately 150 μm.  
     
     
         57 . The apparatus of  claim 49 , wherein the surface area of each pad is approximately 5 μm 2  to approximately 20 μm 2 .  
     
     
         58 . The apparatus of  claim 49 , wherein the surface area of each pad is approximately 10 μm 2 .  
     
     
         59 . An apparatus for processing a plurality of analyses, 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.    
     
     
         60 . The apparatus of  claim 59 , wherein said detection means further comprises signal tracking means for correlating said optical signals from each of said microparticles in each of said digital images to form for each said microparticle of said plurality a sequence of said optical signals.  
     
     
         61 . The apparatus of  claim 60 , wherein said signal tracking means is a CCD camera.  
     
     
         62 . The apparatus of  claim 59 , wherein said analyte is DNA.