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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         30 . A system for determining sequence information at the single molecule level comprising: 
 a detector including a viewing field,    at least one active sequencing complex confined within the viewing field, where the complex comprises: 
 a reaction buffer,  
 a polymerizing agent,  
 a template,  
 a primer adapted to duplex with a portion of the template to form an extendable  
 duplex, and  
 monomer types for the polymerizing agent,  
 where the polymerizing agent and/or the monomer types including tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; and  
   an excitation source adapted to excite the polymerizing agent and/or monomer tags within the viewing field,    where the detector is adapted to detect changes in the detectable properties of the tags before, during and/or after one or a series of monomer incorporation events and to convert the incorporation event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template.    
     
     
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         35 . The system of  claim 30 , wherein the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.  
     
     
         36 . The system of  claim 30 , further comprising a plurality of active complexes confined within the viewing field and where the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having a multiple active complexes therein.  
     
     
         37 . The system of  claim 36 , wherein the monomer types include tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.  
     
     
         38 . The system of  claim 37 , wherein the changes in detectable properties are duration of fluorescence, intensity of fluorescence and/or frequency of fluorescence.  
     
     
         39 . The system of  claim 37 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.  
     
     
         40 . The system of  claim 39 , wherein the FRET acceptor fluorescence occurs when the donor and acceptor are in a close proximity between 100 Å and 100 Å.  
     
     
         41 . The system of  claim 40 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tags are brought into close proximity.  
     
     
         42 . The system of  claim 41 , wherein the close proximity is less than or equal to 50 Å.  
     
     
         43 . The system of  claim 41 , wherein the close proximity is less than or equal to 25 Å.  
     
     
         44 . The system of  claim 41 , wherein the close proximity is less than or equal to 15 Å.  
     
     
         45 . The system of  claim 41 , wherein the close proximity is less than or equal to 10 Å.  
     
     
         46 . The system of  claim 36 , wherein both the polymerizing agent and the monomer types include tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.  
     
     
         47 . The system of  claim 46 , wherein the changes in detectable properties are duration of fluorescence, intensity of fluorescence and/or frequency of fluorescence.  
     
     
         48 . The system of  claim 46 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.  
     
     
         49 . The system of  claim 48 , wherein the FRET acceptor fluorescence occurs when the donor and acceptor are in a close proximity between 100 Å and 10 Å.  
     
     
         50 . The system of  claim 49 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tags are brought into close proximity.  
     
     
         51 . The system of  claim 50 , wherein the close proximity is less than or equal to 50 Å.  
     
     
         52 . The system of  claim 50 , wherein the close proximity is less than or equal to 25 Å.  
     
     
         53 . The system of  claim 50 , wherein the close proximity is less than or equal to 15 Å.  
     
     
         54 . The system of  claim 50 , wherein the close proximity is less than or equal to 10 Å.  
     
     
         55 . An apparatus for determining sequence information at the single molecule level comprising: 
 a substrate,    a detector including a viewing field focused on a region of the substrate,    at least one active sequencing complex confined within the viewing field and comprising a reaction buffer, a polymerizing agent, a template, a primer and monomer types for the polymerizing agent, where the polymerizing agent and/or the monomer types include tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; and    an excitation source adapted to excite the tags within the viewing field,    where the detector is adapted to detect changes in the detectable properties of the tags before, during and/or after one monomer incorporation event or a series of monomer incorporation events and to convert the event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template.    
     
     
         56 . The apparatus of  claim 55 , wherein the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.  
     
     
         57 . The apparatus of  claim 55 , further comprising a plurality of active complexes confined within the viewing field and where the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having multiple active complexes therein.  
     
     
         58 . The apparatus of  claim 57 , wherein the polymerizing agent and/or monomer types including tags and where the tags are all different and where the detectable properties are fluorescence from each tag.  
     
     
         59 . The apparatus of  claim 58 , wherein the changes in detectable properties are duration of fluorescence, intensity of fluorescence and/or frequency of fluorescence.  
     
     
         60 . The apparatus of  claim 58 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.  
     
     
         61 . The apparatus of  claim 60 , wherein the FRET acceptor fluorescence occurs when the donor and acceptor are in a close proximity between 100 Å and 10 Å.  
     
     
         62 . The apparatus of  claim 61 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tag are brought into close proximity.  
     
     
         63 . The apparatus of  claim 62 , wherein the close proximity is less than or equal to 50 Å.  
     
     
         64 . The apparatus of  claim 62 , wherein the close proximity is less than or equal to 25 Å.  
     
     
         65 . The apparatus of  claim 62 , wherein the close proximity is less than or equal to 15 Å.  
     
     
         66 . The apparatus of  claim 62 , wherein the close proximity is less than or equal to 10 Å.  
     
     
         67 . A method for determining sequence information at the single molecule level comprising: 
 providing a detector including a viewing field;    providing at least one active sequencing complex confined within the viewing field, where the complex comprises: 
 a reaction buffer,  
 a polymerizing agent,  
 a template,  
 a primer adapted to duplex with a portion of the template to form an extendable duplex, and  
 monomer types for the polymerizing agent,  
 where the polymerizing agent and/or the monomer types include tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different;  
 an excitation source adapted to excite the tags within the viewing field,  
   detecting changes in the detectable properties of the tags before, during and/or after one monomer incorporation event or a series of monomer incorporation events; and    converting the event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template.    
     
     
         68 . The method of  claim 67 , wherein the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.  
     
     
         69 . The method of  claim 67 , further comprising a plurality of active complexes confined within the viewing field and where the detector further includes data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having multiple active complexes therein and where a spacing between active complexes is such that a single active complex is located within a single data channel or pixel region.  
     
     
         70 . The method of  claim 69 , wherein the polymerizing agent and/or monomer types include tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.  
     
     
         71 . The method of  claim 70 , wherein the changes in detectable properties are duration of fluorescence, intensity of fluorescence and/or frequency of fluorescence.  
     
     
         72 . The method of  claim 70 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.  
     
     
         73 . The method of  claim 72 , wherein the FRET acceptor fluorescence occurs when the donor and acceptor are in a close proximity between 100 Å and 10 Å.  
     
     
         74 . The method of  claim 73 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tag are brought into close proximity.  
     
     
         75 . The method of  claim 74 , wherein the close proximity is less than or equal to 50 Å.  
     
     
         76 . The method of  claim 74 , wherein the close proximity is less than or equal to 25 Å.  
     
     
         77 . The method of  claim 74 , wherein the close proximity is less than or equal to 15 Å.  
     
     
         78 . The method of  claim 74 , wherein the close proximity is less than or equal to 10 Å.