Methods for sequence determination

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

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         32 . A method of sequencing an individual nucleic acid molecule or numerous individual molecules in parallel comprising the steps of: 
 immobilizing a member of the replication complex comprising a polymerizing agent including a polymerizing agent tag a primer and a nucleic acid template sufficiently spaced apart to allow resolvable detection of one or more complexes on a solid support;    contacting the immobilized member with the other members to form bound replication complexes;    incubating the bound replication complexes with monomer for the polymerizing agent, where at least one monomer type includes monomer tag covalently bonded to a portion of the monomer that is released when the tagged monomer is incorporated;    detecting with a detector a change in a detectable property of at least one of the tags during one monomer incorporation event or a plurality of monomer incorporation events; and    relating the changes in the detectable property to an identity of the incorporated monomer or identities of the incorporated plurality of monomer and correlating the identities to one or a sequence of nucleoside in an unknown nucleic acid.    
     
     
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         35 . The method of  claim 32 , wherein the member is the polymerizing agent.  
     
     
         36 . The method of  claim 35 , wherein the polymerizing agent is attached to the support via a molecular tether or linking group.  
     
     
         37 . The method of  claim 36 , wherein the tether or linking group is bonded to a site on the surface of the support and to a site on the polymerizing agent or to a site on a molecule strongly associated with the polymerizing agent.  
     
     
         38 . The method of  claim 37 , wherein the bonded comprises chemical and/or physical interactions sufficient to maintain the polymerizing agent within a given region of the support under normal polymerizing conditions.  
     
     
         39 . The method of  claim 38 , wherein the chemical and/or physical interactions include covalent bonding, ionic bonding, hydrogen bonding, a polar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto to maintain the polymerizing agent in a desired region of the substrate.  
     
     
         40 . The method of  claim 32 , further comprising the step of: 
 prior to contacting, placing the support including the immobilized, tagged polymerizing agents container.    
     
     
         41 . The method of  claim 35 , wherein the polymerizing agent is tethered or anchored on or within a region, area, well, groove, channel or other similar structure on the support capable of being filled with an appropriate polymerizing medium.  
     
     
         42 . The method of  claim 32 , wherein the support is subdivided into a plurality of polymerizing agent structures each having at least one tethered polymerizing agent, where each structure.  
     
     
         43 . The method of  claim 35 , wherein the polymerizing agent is attached to the support to increase and/or maximize data collection by controlling a spacing between members on the support.  
     
     
         44 . The method of  claim 43 , wherein the spacing is such that only a single polymerase is localized within each data collection channel or pixel region within a viewing field of the detector.  
     
     
         45 . The method of  claim 44 , wherein the detector is an imaging system.  
     
     
         46 . The method of  claim 32 , wherein the immobilization is accomplished by anchoring a capture agent or chemically attaching a linking group to the support.  
     
     
         47 . The method of  claim 46 , wherein the capture or linking agents are spaced to useful distances on the support by choosing inherently large capture agents, by conjugating them with or bonding them to molecules which enhance their steric bulk or electrostatic repulsion bulk, or by immobilizing them under conditions chosen to maximize repulsion between polymerizing molecular assembly.  
     
     
         48 . The method of  claim 32 , wherein the member is covalent and/or non-covalent attached to, in or on a surface of the support.  
     
     
         49 . The method of  claim 48 , wherein the surface is an organic surface, an inorganic surface, a nanotube, a similar nano-structure or a porous matrix.  
     
     
         50 . The method of  claim 32 , wherein the polymerizing agent tag is at or near a site on the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent.  
     
     
         51 . The method of  claim 50 , wherein the tag is associated with a protein or probe associated with the polymerizing agent.