Sequence determination using multiply tagged polymerizing agents

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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         20 . A composition comprising a polymerase or reverse transcriptase including a pair of tags, an unknown nucleic acid template, a primer adapted of hybridize with a complementary sequence of a portion of the template to form a primer-template duplex, and monomers for the polymerase or reverse transcriptase, where a detectable property of at least one of the tags undergoes a change before, during and/or after each of a sequence of monomer incorporations.  
     
     
         21 . The composition of  claim 20 , wherein the detectable property has a first value when the polymerase or reverse transcriptase is in a first state and a second value when the polymerase or reverse transcriptase is in a second state, and where the polymerase or reverse transcriptase changes from the first state to the second state and back again during each of the sequence of monomer incorporations.  
     
     
         22 . The composition of  claim 21 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from  E. coli  DNA polymerase I.  
     
     
         23 . The composition of  claim 21 , wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.  
     
     
         24 . The composition of  claim 22 , wherein the polymerase comprises Taq DNA polymerase I having a at least one of the tags attached at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661.  
     
     
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         35 . The composition of  claim 20 , wherein the tags are located at or near the polymerase or reverse transcriptase, associated with the polymerase or reverse transcriptase or covalently bonded to sites on the polymerase or reverse transcriptase.  
     
     
         36 . The composition of  claim 20 , wherein at least one monomer type includes a first monomer tag covalently bonded to a site thereof.  
     
     
         37 . The composition of  claim 36 , wherein the first monomer tag undergoes a change in a monomer tag detectable property before, during or after each incorporation of the at least one monomer type.  
     
     
         38 . The composition of  claim 37 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the monomer type.  
     
     
         39 . The composition of  claim 36 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the at least one monomer type and the tag on the pyrophosphate interacts with at least one of the polymerase or reverse transcriptase tags as it is being released after monomer incorporation.  
     
     
         40 . The composition of  claim 20 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof and a second monomer type includes a second monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         41 . The composition of  claim 40 , wherein the first monomer and second monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first and second monomer types.  
     
     
         42 . The composition of  claim 41 , wherein the first monomer and second monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         43 . The composition of  claim 20 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, and a third monomer type includes a third monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         44 . The composition of  claim 43 , wherein the first, second and third monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second and third monomer types.  
     
     
         45 . The composition of  claim 44 , wherein the first, second and third monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         46 . The composition of  claim 20 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, a third monomer type includes a third monomer tag covalently bonded to a site thereof, and a fourth monomer type includes a fourth monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         47 . The composition of  claim 46 , wherein the first, second, third and fourth monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second, third and fourth monomer types.  
     
     
         48 . The composition of  claim 47 , wherein the first, second, third and fourth monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         49 . A composition comprising a polymerizing agent including at least one pair of tags, a nucleic acid template, a primer adapted to hybridize with a complementary sequence of the nucleic acid to form a primer-template duplex, and monomers for the polymerizing agent, where at least one of the tags in each pair has a detectable property that undergoes a change before, during and/or after each of a sequence of monomer incorporations.  
     
     
         50 . The composition of  claim 49 , wherein the polymerizing agents is any polymerizing agent that polymerizes monomers relative to a specific template in a stepwise fashion.  
     
     
         51 . The composition of  claim 49 , wherein the polymerizing agent is selected from the group consisting of a DNA polymerase, an RNA polymerase, or a reverse transcriptase.  
     
     
         52 . The composition of  claim 49 , wherein the tags are located at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to sites on the polymerizing agent.  
     
     
         53 . The composition of  claim 49 , wherein at least one monomer type includes a first monomer tag covalently bonded to a site thereof.  
     
     
         54 . The composition of  claim 53 , wherein the first monomer tag undergoes a change in a monomer tag detectable property before, during or after each incorporation of the at least one monomer type.  
     
     
         55 . The composition of  claim 54 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the monomer type.  
     
     
         56 . The composition of  claim 54 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the monomer type and the tag on the pyrophosphate interacts with at least one of the polymerase or reverse transcriptase tags as it is being released after monomer incorporation.  
     
     
         57 . The composition of  claim 49 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof and a second monomer type includes a second monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         58 . The composition of  claim 57 , wherein the first monomer and second monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first and second monomer types.  
     
     
         59 . The composition of  claim 58 , wherein the first monomer and second monomer tags are covalently bonded to the P, y or terminal phosphate of their respective monomer types.  
     
     
         60 . The composition of  claim 49 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, and a third monomer type includes a third monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         61 . The composition of  claim 60 , wherein the first, second and third monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second and third monomer types.  
     
     
         62 . The composition of  claim 61 , wherein the first, second and third monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         63 . The composition of  claim 49 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, a third monomer type includes a third monomer tag covalently bonded to a site thereof, and a fourth monomer type includes a fourth monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         64 . The composition of  claim 63 , wherein the first, second, third and fourth monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second, third and fourth monomer types.  
     
     
         65 . The composition of  claim 64 , wherein the first, second, third and fourth monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         66 . A composition comprising a polymerizing agent including at least one pair of tags, a nucleic acid template, a primer adapted to hybridize with a complementary sequence of the nucleic acid to form a primer-template duplex, and monomers for the polymerizing agent, where the tags are substantially inactive when the polymerizing agent is in a first state and substantially active when the polymerizing agent is in a second state and where the polymerizing agent transition from the first state to the second state during each of a sequence of monomer incorporations.  
     
     
         67 . The composition of  claim 66 , wherein the polymerizing agents is any polymerizing agent that polymerizes monomers relative to a specific template in a stepwise fashion.  
     
     
         68 . The composition of  claim 66 , wherein the polymerizing agent is selected from the group consisting of a DNA polymerase, an RNA polymerase, or a reverse transcriptase.  
     
     
         69 . The composition of  claim 66 , wherein the tags are located at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to sites on the polymerizing agent.  
     
     
         70 . The composition of  claim 66 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof.  
     
     
         71 . The composition of  claim 70 , wherein the first monomer tag undergoes a change in a monomer tag detectable property before, during or after each incorporation of the first monomer type.  
     
     
         72 . The composition of  claim 71 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the monomer type.  
     
     
         73 . The composition of  claim 71 , wherein the first monomer tag is covalently bonded to the β, γ or terminal phosphate of the monomer type and the tag on the pyrophosphate interacts with at least one of the polymerase or reverse transcriptase tags as it is being released after monomer incorporation.  
     
     
         74 . The composition of  claim 66 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof and a second monomer type includes a second monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         75 . The composition of  claim 74 , wherein the first monomer and second monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first and second monomer types.  
     
     
         76 . The composition of  claim 75 , wherein the first monomer and second monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         77 . The composition of  claim 66 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, and a third monomer type includes a third monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         78 . The composition of  claim 77 , wherein the first, second and third monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second and third monomer types.  
     
     
         79 . The composition of  claim 78 , wherein the first, second and third monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.  
     
     
         80 . The composition of  claim 66 , wherein a first monomer type includes a first monomer tag covalently bonded to a site thereof, a second monomer type includes a second monomer tag covalently bonded to a site thereof, a third monomer type includes a third monomer tag covalently bonded to a site thereof, and a fourth monomer type includes a fourth monomer tag covalently bonded to a site thereof, where the monomer tags are the same or different.  
     
     
         81 . The composition of  claim 80 , wherein the first, second, third and fourth monomer tags undergo a change in a monomer tag detectable property before, during or after each incorporation of the first, second, third and fourth monomer types.  
     
     
         82 . The composition of  claim 81 , wherein the first, second, third and fourth monomer tags are covalently bonded to the β, γ or terminal phosphate of their respective monomer types.