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.

1 . A method of sequencing a nucleic acid molecule, comprising:
 immobilizing a member of a replication complex comprising one or more polymerases and one or more target nucleic acid templates on a support to form a bound replication complex;   contacting at least one bound replication complex with one or more tagged nucleotides or tagged nucleotide analogs, wherein the tag has a detectable property and is attached to a portion of the nucleotide or nucleotide analog that is released upon incorporation;   detecting, in real-time or near real time, a change in the detectable property of at least one tag before, during or after incorporation of one or more tagged nucleotides or nucleotide analogs, where the incorporation results in the release of the tag; and   relating the detected change in the detectable property of the at least one tag to an identity of one or more incorporated nucleotides or nucleotide analogs.   
     
     
         2 . The method of  claim 1 , wherein the immobilized member is a polymerase. 
     
     
         3 . The method of  claim 2 , wherein the polymerase is attached to the support via a molecular tether or linking group. 
     
     
         4 . The method of  claim 3 , wherein the tether or linking group is bonded to a site on the support and to a site on the polymerase or to a site on a molecule strongly associated with the polymerase. 
     
     
         5 . The method of  claim 4 , wherein the bonding involves covalent bonding interactions, ionic bonding interactions, hydrogen bonding interactions, apolar bonding interactions, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto to maintain the polymerase in the desired region of the support. 
     
     
         6 . The method of  claim 2 , wherein the polymerase 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. 
     
     
         7 . The method of  claim 2 , wherein the support is subdivided into a plurality of structures, each structure including at least one bound replication complex bound thereon or therein. 
     
     
         8 . The method of  claim 2 , wherein the polymerase is directly or indirectly attached to the support and the spacing between immobilized of each replication complex is controlled. 
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . The method of  claim 9 , wherein the detector is an imaging system. 
     
     
         11 . The method of  claim 2 , wherein the immobilization is accomplished by via anchoring a capture agent or chemically attaching a linking group to the support and attaching the polymerase thereto. 
     
     
         12 . The method of  claim 11 , 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, and immobilizing them under conditions chosen to maximize repulsion between replication complexes. 
     
     
         13 . The method of  claim 1 , wherein the member is covalently and/or non-covalently attached to, in or on a surface of the support. 
     
     
         14 . The method of  claim 13 , wherein the surface is an organic surface, an inorganic surface, a nanotube, a similar nano-structure or a porous matrix. 
     
     
         15 . The method of  claim 2 , wherein the polymerase comprises a detectable polymerase tag located at or near a site on the polymerase, associated with polymerase or covalently bonded to a site on the polymerase. 
     
     
         16 . The method of  claim 15 , wherein the polymerase tag is associated with a protein or probe associated with the polymerase. 
     
     
         17 . The method of  claim 15 , wherein the change in detectable property is based on an interaction between the tag of an incorporating tagged nucleotide or nucleotide analog and the polymerase tag. 
     
     
         18 . The method of  claim 15 , wherein the tag of an incorporating tagged nucleotide or nucleotide analog and the polymerase tag form a fluorescence resonance energy transfer (FRET) pair. 
     
     
         19 . The method of  claim 18 , wherein one or more changes in the detectable property of a tag is based on a FRET interaction between the tag of an incorporating tagged nucleotide or nucleotide analog and the polymerase tag. 
     
     
         20 . The method of  claim 1 , further comprising:
 correlating the identity of one or more nucleotides or nucleotide analogs to a sequence of the nucleic acid template.   
     
     
         21 . A method for sequencing single nucleic acid molecules in real-time or near real-time, comprising:
 providing a composition comprising a target nucleic acid template, a tagged polymerase, and one or more types of nucleotides or nucleotide analogs, where at least one type of nucleotide or nucleotide analog comprises tagged nucleotides or tagged nucleotide analogs comprising a first nucleotide tag bonded to a part of the nucleotide or nucleotide analog that is released due to action of the polymerase during nucleotide incorporation, where the polymerase or template is immobilized on a support,   detecting a time sequence of tagged nucleotide or tagged nucleotide analog incorporation events in real-time or near real-time, where each event comprises a change in a detectable property of the tag of a tagged nucleotide or nucleotide analog type upon incorporation; and   relating the detected change in the detectable property of the at least one tag to an identity of one or more incorporated nucleotides or nucleotide analogs.   
     
     
         21 . The method of  claim 20 , further comprising: identifying one or more tagged nucleotide or tagged nucleotide analog incorporation events to produce a partial or complete sequence of the template. 
     
     
         22 . The method of  claim 20 , wherein a second nucleotide or nucleotide analog type comprises a second tagged nucleotide or second tagged nucleotide analog type comprising a second nucleotide tag bonded to a part of the nucleotide or nucleotide analog that is released due to action of the polymerase during nucleotide incorporation, and wherein the first nucleotide tag and the second nucleotide tag are different. 
     
     
         23 . The method of  claim 22 , further comprising: identifying one or more tagged nucleotide or tagged nucleotide analog incorporation events to produce a partial or complete sequence of the template.