Methods and apparatuses for analyzing polynucleotide sequences

Abstract

Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

1 . A method of conducting single molecule sequencing, the method comprising:
 providing a device comprising a synthesis channel or a reaction chamber,   introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;   conducting said reaction,   determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,   wherein one or more of the reagents is introduced in the synthesis channel or reaction chamber at a flow rate that does not cause substantial sheering of nucleic acid template and/or dislocation of the polymerase from nucleic acid complexes.   
     
     
         2 . A method of conducting single molecule sequencing, the method comprising:
 providing a device comprising a synthesis channel or a reaction chamber,   introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;   conducting said reaction,   determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,   wherein one or more of the reagents is introduced in the synthesis channel or reaction chamber at a flow rate that maintains substantially laminar flow in the channel or chamber.   
     
     
         3 . A method of conducting single molecule sequencing, the method comprising:
 providing a device comprising a synthesis channel or a reaction chamber,   introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;   conducting said reaction,   determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,   wherein one or more of the reagents is introduced into the synthesis channel or reaction chamber at a flow rate below 10 cm/sec.   
     
     
         4 . The method of  claim 3 , wherein the flow rate is chosen from 0.1-1 cm/sec and 1-10 cm/sec. 
     
     
         5 . The method of  claim 4 , wherein the flow rate is 0.1-1 cm/sec. 
     
     
         6 . The method of  claim 1 , wherein the template is at least 1 kb in length. 
     
     
         7 . The method of  claim 1 , wherein the template is at least about 20 kb in length. 
     
     
         8 . The method of  claim 2 , wherein the template is at least 1 kb in length. 
     
     
         9 . The method of  claim 2 , wherein the template is at least about 20 kb in length. 
     
     
         10 . The method of  claim 3 , wherein the template is at least 1 kb in length. 
     
     
         11 . The method of  claim 3 , wherein the template is at least about 20 kb in length. 
     
     
         12 . The method of  claim 1 , further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate that does not cause substantial sheering of nucleic acid templates and/or dislocation of the polymerase from nucleic acid complexes. 
     
     
         13 . The method of  claim 2 , further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate that maintains substantially laminar flow in the channel or chamber. 
     
     
         14 . The method of  claim 3 , further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate below 10 cm/sec. 
     
     
         15 . The method of  claim 14 , wherein the flow rate is chosen from 0.1-1 cm/sec and 1-10 cm/sec. 
     
     
         16 . The method of  claim 15 , wherein the flow rate is 0.1-1 cm/sec. 
     
     
         17 . The method of  claim 1 , wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera. 
     
     
         18 . The method of  claim 2 , wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera. 
     
     
         19 . The method of  claim 3 , wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera. 
     
     
         20 . The method of  claim 1 , wherein the dye-labeled nucleotides are fluorescently labeled.