Methods of Amplifying and Sequencing Nucleic Acids

Abstract

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

1 ) A system for sequencing nucleic acids, comprising:
 a perfusion chamber comprising an upper side and an array of at least 10,000 reaction chambers a plurality of which comprise a bead each with multiple clonal copies of a species of nucleic acid immobilized thereon, wherein the sealed perfusion chamber further comprises a first port detachably coupled to a fluidics subsystem that comprises a single inlet conduit operatively coupled to a manifold, and an A nucleotide tube, a G nucleotide tube, a T nucleotide tube, and a C nucleotide tube.   
     
     
         2 ) The system of  claim 1 , wherein:
 the perfusion chamber further comprises a second port to withdraw reagents and reaction products.   
     
     
         3 ) The system of  claim 1 , wherein:
 the multiple clonal copies comprise at least 100,000 copies.   
     
     
         4 ) The system of  claim 1 , wherein:
 the inlet conduit utilizes a pressurized system to drive a positive flow of fluids through the sealed perfusion chamber.   
     
     
         5 ) The system of  claim 4 , wherein:
 the positive flow comprises a flow rate in a range from 0.5 to 50 ml/min.   
     
     
         6 ) The system of  claim 1 , wherein:
 the manifold further comprises a wash tube.   
     
     
         7 ) The system of  claim 1 , wherein:
 the manifold further comprises a polymerase tube.   
     
     
         8 ) The system of  claim 1 , wherein:
 the perfusion chamber comprises a plastic housing positioned over the array of reaction chambers.   
     
     
         9 ) The system of  claim 1 , wherein:
 the upper side of the perfusion chamber is transparent.   
     
     
         10 ) The system of  claim 1 , wherein:
 the perfusion chamber is sealed.   
     
     
         11 ) The system of  claim 1 , wherein:
 the perfusion chamber comprises a ceiling positioned about 300 μm over the array of reaction chambers.   
     
     
         12 ) The system of  claim 1 , wherein:
 the reaction chambers are circular, cylindrical, or multisided.   
     
     
         13 ) The system of  claim 12 , wherein:
 the multisided reaction chambers are hexagonal.   
     
     
         14 ) The system of  claim 1 , wherein:
 the reaction chambers comprise a smooth wall surface and a planar bottom surface.   
     
     
         15 ) The system of  claim 1 , wherein:
 the clonal copies of the species of nucleic acid are immobilized on the bead by a covalent linkage.   
     
     
         16 ) The system of  claim 1 , wherein:
 the clonal copies of the species of nucleic acid comprise an oligonucleotide primer covalently linked to the bead and a complimentary copy of a template nucleic acid extended therefrom.   
     
     
         17 ) The system of  claim 1 , wherein:
 the clonal copies of the species of nucleic acid comprise a detectable sequence tag associated with a biological source.   
     
     
         18 ) The system of  claim 1 , wherein:
 the bead comprises an agarose, acrylamide, or polyacrylamide material.   
     
     
         19 ) The system of  claim 1 , wherein:
 the array comprises a surface coating that comprises a self-assembled monolayer or a metal layer.   
     
     
         20 ) The system of  claim 1 , wherein:
 the array comprises a surface coating that increases the stability of linked macromolecules.   
     
     
         21 ) The system of  claim 1 , wherein:
 the array comprises a surface coating that minimizes absorption of macromolecules.