Sequence determination in confined regions

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 nucleic acid molecules at the single molecule level comprising the steps of: 
 immobilizing a member of a replication complex comprising a polymerizing agent, an oligonucleotide primer and a nucleic acid template on or in a substrate;    contacting the immobilized member with the non-immobilized members of the replication complex to form an immobilized replication complex;    incubating the immobilized replication complex with monomers for the polymerizing agent, where at least one of the monomer types includes a monomer tag covalently bonded to a site on the monomer that is not incorporated into a growing complementary nucleic acid sequence, where the monomer tag has a detectably property capable of being detected by a detector;    detecting a change in the detectable property of each monomer tag as a tagged monomer is incorporated by the polymerizing agent into the growing complementary nucleic acid sequence; and    converting the detected changes in the detectable property of the monomer tags to an identity of one monomer or a plurality of monomers corresponding to one nucleotide or a plurality of nucleotides of the template.    
     
     
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         35 . The method of  claim 32 , wherein the member comprises the polymerizing agent and the substrate includes a structure and the polymerizing agent is immobilized on or in the structure.  
     
     
         36 . The method of  claim 35 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         37 . The method of  claim 32 , wherein the member comprises the oligonucleotide primer and the substrate includes a structure and the primer is immobilized on or in the structure.  
     
     
         38 . The method of  claim 37 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         39 . The method of  claim 32 , wherein the member comprises the nucleic acid template and the substrate includes a structure and the template is immobilized on or in the structure.  
     
     
         40 . The method of  claim 39 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         41 . The method of  claim 32 , wherein a plurality of members are immobilized in the immobilizing step and a plurality of immobilized replication complexes are formed in the adding step.  
     
     
         42 . The method of  claim 41 , wherein the plurality of immobilized replication complexes are sufficiently spaced apart on or in the substrate to afford independent detection of a portion of the immobilized replication complexes by the detector.  
     
     
         43 . The method of  claim 32 , wherein the polymerizing agent comprises naturally occurring polymerases or reverse transcriptases or mutated naturally occurring polymerases or reverse transcriptases, where the mutations comprise: (a) replacement of one amino acid or a plurality of amino acids with another amino acid or other amino acids, and/or (b) insertion or deletion of one amino acid or a plurality of amino acids, where the polymerizing agents are capable of incorporating the tagged monomers and capable ofbeing isolated and purified in amounts sufficient to facilitate detection of the tagged monomers during monomer incorporation.  
     
     
         44 . The method of  claim 43 , wherein the polymerizing agent lacks 3′ to 5′ exonuclease activity.  
     
     
         45 . The method of  claim 44 , wherein the polymerizing agent comprises an DNA polymerase, an RNA polymerase, a reverse transcriptase or mutants thereof.  
     
     
         46 . The method of  claim 44 , wherein the polymerizing agent comprises a DNA polymerase or mutants thereof.  
     
     
         47 . The method of  claim 44 , wherein the polymerizing agent comprises DNA polymerase I from  T. aquaticus  or  E. coli , Bateriophage T4 DNA polymerase, T7 DNA polymerase, Klenow Fragment, Pfu DNA Polymerase, Sequenase or mutants thereof.  
     
     
         48 . The method of  claim 46 , wherein the DNA polymerase comprises a mutant DNA polymerase including one amino acid or a plurality of amino acids replacements adapted to produce amino acid sites in the polymerase amenable to attaching a polymerase tag.  
     
     
         49 . The method of  claim 48 , wherein the mutant DNA polymerase includes a tag covalently bonded to at least one of the amino acid replacement sites on the polymerase or to at least one naturally occurring amino acid site on the polymerase.  
     
     
         50 . The method of  claim 45 , wherein the polymerase tag is a fluorescent donor and the monomer tag is a fluorescent acceptor, where the donor and acceptor are capable of undergoing fluorescence resonance energy transfer (FRET).  
     
     
         51 . The method of  claim 32 , wherein at least one monomer type further includes a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         52 . The method of  claim 32 , wherein at least two monomer types further include a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         53 . The method of  claim 32 , wherein at least three monomer types further include a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         54 . The method of  claim 32 , wherein each monomer type further includes a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         55 . A method of sequencing nucleic acid molecules comprising the steps of: 
 confining a plurality of polymerizing agents on or in a substrate to form a plurality of confined polymerizing agents;    contacting the confined polymerizing agents with a solution including a nucleic acid template and oligonucleotide primers, where the primers are adapted to duplex with a portion of the templates to form extendable nucleic acid duplexes and the duplexes are adapted to complex with some or all of the confined polymerizing agents to form confined replication complexes;    incubating the confined replication complexes with monomer types for the polymerizing agent, where at least two of the monomer types include unique monomer tags covalently bonded to monomer sites on the monomer types that are not incorporated into a growing complementary nucleic acid sequence and where the monomer tags have a detectable property capable of being detected by a detector;    detecting a change in the detectable property of each monomer tag as a tagged monomer is incorporated by the polymerizing agent into the growing complementary nucleic acid sequence; and    converting the detected changes in the detectable property of the incorporated tagged monomer to an identity of one nucleotide or a plurality of nucleotides of the template.    
     
     
         56 . A method of sequencing nucleic acid molecules comprising the steps of: 
 confined a plurality of polymerizing agents on or in a substrate to form a plurality of confined polymerizing agents, where each polymerizing agent includes a polymerizing agent donor fluorescent tag covalently bonded to a site on the polymerizing agent or associated with a molecule associated with the polymerizing agent;    contacting the confined polymerizing agents with a solution including a nucleic acid template and oligonucleotide primers, where the primers are adapted to duplex with a portion of the template to form extendable nucleic acid duplexes and the duplexes are adapted to complex with some or all of the confined polymerizing agents to form confined replication complexes;    incubating the confined replication complex with four nucleotide types for the polymerizing agent, where a first nucleotide type includes a first acceptor fluorescent tag covalently bonded to a site thereof and a second nucleotide type includes a second acceptor fluorescent tag covalently bonded to a site thereof and where the nucleotide tags are not incorporated by the polymerizing agent into a growing complementary nucleic acid sequence and where the nucleotide tags are capable of undergoing fluorescence resonance energy transfer (FRET) with the donor tag and where the acceptor tags are the same or different;    detecting fluorescent light emitted by each monomer tag as a tagged nucleotide is incorporated by a polymerizing agent into a growing complementary nucleic acid sequence via an FRET interaction between the incorporating acceptor tag on the tagged nucleotide and the donor tag on the polymerizing agent incorporating the tagged monomer to produce data evidencing a sequence of tagged monomer incorporation events; and    converting the FRET data into an identity of one nucleotide or a plurality of nucleotides of the template.    
     
     
         57 . A method of sequencing nucleic acid molecules comprising the steps of: 
 confining a plurality of polymerizing agents on or in a substrate to form a plurality of confined polymerizing agents, where each polymerizing agent includes a polymerizing agent donor fluorescent tag covalently bonded to a site on the polymerizing agent or associated witha molecule associated with the polymerizing agent;    contacting the confined polymerizing agents with a solution including a nucleic acid template and oligonucleotide primers, where the primers are adapted to duplex with a portion of the template to form extendable nucleic acid duplexes and the duplexes are adapted to complex with some or all of the confined polymerizing agents to form confined replication complexes;    incubating the confined replication complex with four dNPT types for the polymerizing agent, where a first monomer type includes a first acceptor fluorescent tag covalently bonded to a site thereof, a second monomer type includes a second acceptor fluorescent tag covalently bonded to a site thereof, and a third monomer type includes a third acceptor fluorescent tag covalently bonded to a site thereof, and where the monomer tags are not incorporated by the polymerizing agent into a growing complementary nucleic acid sequence, and where the monomer tags are capable of undergoing fluorescence resonance energy transfer (FRET) with the donor tag and where the acceptor tags are the same or different;    detecting fluorescent light emitted by each monomer tag as a tagged monomer is incorporated by a polymerizing agent into a growing complementary nucleic acid sequence via an FRET interaction between the incorporating acceptor tag on the monomer and the donor tag on the polymerizing agent incorporating the tagged monomer to produce data evidencing a sequence of monomer incorporation events; and    converting the FRET data into an identity of one nucleotide or a plurality of nucleotides of the template.    
     
     
         58 . A method of sequencing nucleic acid molecules comprising the steps of: 
 confined a plurality of polymerizing agents on or in a substrate to form a plurality of immobilized polymerizing agents, where each polymerizing agent includes a polymerizing agent donor fluorescent tag covalently bonded to a site on the polymerizing agent or associated with a molecule associated with the polymerizing agent;    contacting the confined polymerizing agents with a solution including a nucleic acid template and oligonucleotide primers, where the primers are adapted to duplex with a portion of the template to form extendable nucleic acid duplexes and the duplexes are adapted to complex with some or all of the confined polymerizing agents to form confined replication complexes;    incubating the confined replication complex with four dNTP types for the polymerizing agent, where a first monomer type includes a first acceptor fluorescent tag covalently bonded to a site thereof, a second monomer type includes a second acceptor fluorescent tag covalently bonded to a site thereof, a third monomer type includes a third acceptor fluorescent tag covalently bonded to a site thereof, and a fourth monomer type includes a fourth acceptor fluorescent tag covalently bonded to a site thereof, and where the monomer tags are not incorporated by the polymerizing agent into a growing complementary nucleic acid sequence, and where the monomer tags are capable of undergoing fluorescence resonance energy transfer (FRET) with the donor tag and where the acceptor tags are the same or different;    detecting fluorescent light emitted by each monomer tag as a tagged monomer is incorporated by a polymerizing agent into a growing complementary nucleic acid sequence via an FRET interaction between the incorporating acceptor tag on the monomer and the donor tag on the polymerizing agent incorporating the tagged monomer to produce data evidencing a sequence of monomer incorporation events; and    converting the FRET data into an identity of one nucleotide or a plurality of nucleotides of the template.    
     
     
         59 . A method comprising the steps of: 
 confining a polymerizing agent on or in a substrate;    incubating the polymerizing agent in the presence of a template polymer comprising a sequence of the monomers, optionally primers adapted to duplex with a portion of the template polymer, and monomers for the polymerizing agent, where each monomer type includes an unique monomer tag covalently bonded to a site of the monomer that is not incorporated by the polymerizing agent into a growing complementarypolymer comprising a sequence of the monomers complementary to the template polymer and each monomer tag has a detectably property capable of being detected by a detector;    detecting a change in the detectable property as each monomer is incorporated by the polymerizing agent into the growing complementary polymer; and    converting the detected changes in the detectable property of the monomer tag of each incorporated monomer to a sequence of monomers in the template polymer.    
     
     
         60 . The method of  claim 55 , wherein the polymerizing agent comprises a molecule or molecular assembly.  
     
     
         61 . A method of sequencing nucleic acid molecules at the single molecule level comprising the steps of: 
 confining a member of a replication complex comprising a polymerizing agent, a primer and a template in a region, area, well, groove, channel or other similar structure on the substrate capable of being filled with an appropriate polymerizing medium;    contacting the confined member with the other members of the replication complex to form a confined replication complex;    incubating the confined replication complex with monomers for the polymerizing agent in the presence of the medium, where at least one of the monomer types includes monomer tag attached to a site on the monomer that is not incorporated into a growing complementary sequence of monomers, where the monomer tag has a detectably property capable of being detected by a detector;    detecting a change in the detectable property of each monomer tag as a tagged monomer is incorporated by the polymerizing agent into the growing complementary sequence; and    converting the detected changes in the detectable property to an identity of one monomer or a plurality of monomers corresponding to one nucleotide or a plurality of nucleotides of the template.    
     
     
         62 . The method of  claim 63 , wherein the member comprises the polymerizing agent and the substrate includes a structure and the polymerizing agent is confined on or in the structure.  
     
     
         63 . The method of  claim 64 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         64 . The method of  claim 63 , wherein the member comprises the oligonucleotide primer and the substrate includes a structure and the primer is confined on or in the structure.  
     
     
         65 . The method of  claim 66 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         66 . The method of  claim 63 , wherein the member comprises the nucleic acid template and the substrate includes a structure and the template is confined on or in the structure.  
     
     
         67 . The method of  claim 68 , wherein the structure comprises a region, an area, a well, a groove or a channel.  
     
     
         68 . The method of  claim 63 , wherein a plurality of members are confined in the confining step and a plurality of confined replication complexes are formed in the adding step.  
     
     
         69 . The method of  claim 70 , wherein the plurality of confined replication complexes are sufficiently spaced apart on or in the substrate to afford independent detection of a portion of the confined replication complexes by the detector.  
     
     
         70 . The method of  claim 63 , wherein the polymerizing agent comprises naturally occurring polymerases or reverse transcriptases or mutated naturally occurring polymerases or reverse transcriptases, where the mutations comprise: (a) replacement of one amino acid or a plurality of amino acids with another amino acid or other amino acids, and/or (b) insertion or deletion of one amino acid or a plurality of amino acids, where the polymerizing agents are capable of incorporating the tagged monomers and capable of being isolated and purified in amounts sufficient to facilitate detection of the tagged monomers during monomer incorporation.  
     
     
         71 . The method of  claim 72 , wherein the polymerizing agent lacks 3′ to 5′ exonuclease activity.  
     
     
         72 . The method of  claim 73 , wherein the polymerizing agent comprises an DNA polymerase, an RNA polymerase, a reverse transcriptase or mutants thereof.  
     
     
         73 . The method of  claim 73 , wherein the polymerizing agent comprises a DNA polymerase or mutants thereof.  
     
     
         74 . The method of  claim 73 , wherein the polymerizing agent comprises DNA polymerase I from  T. aquaticus  or  E. coli , Bateriophage T4 DNA polymerase, T7 DNA polymerase, Klenow Fragment, Pfu DNA Polymerase, Sequenase or mutants thereof.  
     
     
         75 . The method of  claim 76 , wherein the DNA polymerase comprises a mutant DNA polymerase including one amino acid or a plurality of amino acids replacements adapted to produce amino acid sites in the polymerase amenable to attaching a polymerase tag.  
     
     
         76 . The method of  claim 77 , wherein the mutant DNA polymerase includes a tag covalently bonded to at least one of the amino acid replacement sites on the polymerase.  
     
     
         77 . The method of  claim 78 , wherein the polymerase tag is a fluorescent donor and the monomer tag is a fluorescent acceptor, where the donor and acceptor are capable of undergoing fluorescence resonance energy transfer (FRET).  
     
     
         78 . The method of  claim 63 , wherein at least one monomer type further includes a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         79 . The method of  claim 63 , wherein at least two monomer types further include a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         80 . The method of  claim 63 , wherein at least three monomer types further include a second tag attached to a site of the monomer that remains with the monomer after incorporation.  
     
     
         81 . The method of  claim 63 , wherein each monomer type further includes a second tag attached to a site of the monomer that remains with the monomer after incorporation.