Tagged monomers for use in 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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         33 . A composition comprising a monomer capable of being incorporated into a growing molecular chain by a polymerizing agent, where the monomer includes a tag selected from the group consisting of a fluorescent tag, an NMR active tag, an IR active tag, an UV active tag, and a far UV active tag.  
     
     
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         35 . The composition of  claim 33 , wherein the first tag is an NMR active.  
     
     
         36 . The composition of  claim 33 , wherein the monomer is a naturally occurring or synthetic nucleotide and the first tag is an NMR active atom.  
     
     
         37 . The composition of  claim 33 , wherein the first tag is covalently bonded to a site on the monomer that is released during incorporation of the monomer into the growing molecular chain.  
     
     
         38 . The composition of  claim 33 , wherein the monomer is a naturally occurring or synthetic nucleotide and the first tag is covalently bonded to a site on the nucleotide that is released during incorporation of the nucleotide into the growing molecular chain.  
     
     
         39 . The composition of  claim 38 , wherein the first tag comprises a fluorescent tag covalently bonded to a terminal phosphate, an γ-phosphate, or an β-phosphate of the nucleotide.  
     
     
         40 . A composition comprising a monomer including a first tag and a second tag, where the monomer is capable of being incorporated into a growing molecular chain by a polymerizing agent and at least one of the tags has a detectable property.  
     
     
         41 . The composition of  claim 40 , wherein the first and second tags are different.  
     
     
         42 . The composition of  claim 40 , wherein the first tag is an NMR active atom associated with a portion of the monomer incorporated into the growing molecular chain.  
     
     
         43 . The composition of  claim 40 , wherein the first tag is an NMR active atom associated with a portion of the monomer released during monomer incorporation into the growing molecular chain.  
     
     
         44 . The composition of  claim 40 , wherein the first and second tags are different and each tag has a detectable property.  
     
     
         45 . The composition of  claim 40 , wherein the first tag remains with a portion of the monomer incorporated into the growing molecular chain, a persistent tag, and the second tag remains with a portion of the monomer released during monomer incorporation, a non-persistent tag.  
     
     
         46 . The composition of  claim 45 , wherein the monomer is a naturally occurring or synthetic nucleotide.  
     
     
         47 . The composition of  claim 45 , wherein the first or persistent tag is on an α-phosphate, a sugar moiety, or a base moiety of the nucleotide and the second or non-persistent tag is on a terminal phosphate, an γ-phosphate, or P-phosphate of the nucleotide.  
     
     
         48 . The composition of  claim 45 , wherein the first or persistent tag is covalently bonded to an α-phosphate, a sugar moiety, or a base moiety of the nucleotide and the second or non-persistent tag is covalently bonded to a terminal phosphate, an γ-phosphate, or β-phosphate of the nucleotide.  
     
     
         49 . The composition of  claim 48 , wherein the first and second tags comprise molecular tags selected from the group consisting of fluorescent tags, NMR active tags, IR active tags, an UV active tags, and far UV active tags.  
     
     
         50 . The composition of  claim 46 , wherein the first tag comprises a molecular fluorescent tag, where first fluorescent tag is covalently boned to a terminal phosphate, an γ-phosphate or an β-phosphate of the nucleotide and the second fluorescent tag is on an α-phosphate, a sugar moiety, or a base moiety of the nucleotide.  
     
     
         51 . A composition comprising a monomer capable of being incorporated into a growing molecular chain by a polymerase, where the compound includes a first tag having a detectable property and covalently bonded to a site on the monomer that is released during incorporation of the monomer into the growing molecular chain.  
     
     
         52 . The composition of  claim 51 , wherein the first tag comprises a molecular tag selected from the group consisting of a fluorescent tag, an NMR active tag, an IR active tag, an UV active tag, and a far UV active tag.  
     
     
         53 . The composition of  claim 51 , wherein the monomer comprising a naturally occurring or synthetic nucleotide and the first tag is selected from the group consisting of a fluorescent tag, an NMR active tag, an IR active tag, an UV active tag, and a far UV active tag.  
     
     
         54 . The composition of  claim 53 , wherein the first tag comprises a fluorescent tag.  
     
     
         55 . The composition of  claim 54 , wherein the first tag is covalently bonded to a terminal phosphate, an γ-phosphate, or β-phosphate of the nucleotide.  
     
     
         56 . The composition of  claim 55 , wherein the first tag is covalently bonded to a terminal phosphate.  
     
     
         57 . The composition of  claim 55 , wherein the first tag is covalently bonded to an γ-phosphate.  
     
     
         58 . The composition of  claim 55 , wherein the first tag is covalently bonded to β-phosphate.  
     
     
         59 . A composition comprising a naturally occurring or synthetic nucleotide including a tag covalently bonded to the γ-phosphate, where the tag has a detectable property.  
     
     
         60 . The composition of  claim 59 , wherein the tag is selected from the group consisting of fluorescent dyes, NMR active groups, IR active tags, far IR active tags, UV active tags, and far UV active tags.  
     
     
         61 . The composition of  claim 60 , wherein the molecular tag comprises a fluorescent tag.  
     
     
         62 . A composition comprising a naturally occurring or synthetic nucleotide including a tag covalently bonded to the γ-phosphate via a linker, where the tag has a detectable property.  
     
     
         63 . The composition of  claim 62 , wherein the modified nucleotide is a compound having the general formula:  
         FR-L-Z-γ-phosphate β-phosphate-α-phosphate-Sugar-Base  
       where 
 FR is a molecular fluorescent tag,  
 L is a linker group,  
 Z is selected from the group consisting of O, NH, NR, CO 2 , SiO, and GeO, where R is an alkyl, aryl, aralkyl, alkaryl, halogenated analogs thereof or hetero atom analogs thereof,  
 Sugar is a five carbon sugar moiety, and  
 Base is selected from the group consisting of A, C, G, T, and U.  
 
     
     
         64 . The composition of  claim 63 , wherein the L is 1-hydroxy-6-amino hexane.  
     
     
         65 . The composition of  claim 60 , wherein the tag is selected from the group consisting of a fluorescent tag, an NMR active tag, an IR active tag, a far IR active tag, an UV active tag, and a far UV active tag.  
     
     
         66 . The composition of  claim 65 , wherein the tag comprises a fluorescent tag.  
     
     
         67 . A composition comprising a naturally occurring or synthetic nucleotide including a tag covalently bonded to the P-phosphate, where the tag has a detectable property.  
     
     
         68 . The composition of  claim 67 , wherein the tag is selected from the group consisting of fluorescent dyes, NMR active groups, IR active tags, far IR active tags, UV active tags, and far UV active tags.  
     
     
         69 . The composition of  claim 68 , wherein the tag comprises a fluorescent tag.  
     
     
         70 . A composition comprising a naturally occurring or synthetic nucleotide including a molecular tag covalently bonded to the β-phosphate via a linker, where the molecular tag has a detectable property.  
     
     
         71 . The composition of  claim 70 , wherein the modified nucleoside is a compound having the general formula:  
         γ-phosphate-β-phosphate(FR-L-Z)-α-phosphate-Sugar-Base  
       where FR is a fluorescent molecular tag, L is a linker group, Z O, NH, NR, CO 2 , SiO, GeO, where R is an alkyl, aryl, aralkyl, alkaryl, halogenated analogs thereof or hetero atom analogs thereof, Sugar is a five carbon atom sugar, and Base is selected from the group consisting of A, C, G, T, or U.  
     
     
         72 . The composition of  claim 71 , wherein the L is 1-hydroxy-6-amino hexane.  
     
     
         73 . The composition of  claim 72 , wherein the molecular tag is selected from the group consisting of fluorescent dyes, NMR active groups, IR active tags, far IR active tags, UV active tags, and far UV active tags.  
     
     
         74 . The composition of  claim 73 , wherein the molecular tag comprises a fluorescent tag.  
     
     
         75 . A modified nucleotide composition comprising a naturally occurring or synthetic nucleotide including two tags on different sites of the nucleotide, where at least one tag has a detectable property.  
     
     
         76 . The composition of  claim 75 , wherein the first tag is on the α-phosphate, a site on the sugar moiety, or a site on the base moiety of the nucleotide and the second tag is covalently bonded to the β-phosphate, the γ-phosphate or the terminal phosphate.  
     
     
         77 . The composition of  claim 76 , wherein the second tag has a detectable property.  
     
     
         78 . The composition of  claim 77 , wherein the second tag is selected from the group consisting of a fluorescent tag, an NMR active tag, an IR active tag, an UV active tag, and a far UV active tag.  
     
     
         79 . The composition of  claim 78 , wherein the tags comprise fluorescent tags.  
     
     
         80 . The composition of  claim 79 , wherein the first tag is covalently bonded to the α-phosphate, a site on the sugar moiety, or a site on the base through a linker group.  
     
     
         81 . The composition of  claim 79 , wherein the second tag comprises a fluorescent tag covalently bonded to the β-phosphate, the γ-phosphate or the terminal phosphate through a linker group.  
     
     
         82 . The composition of  claim 79 , wherein the first tag is covalently bonded to the α-phosphate, a site on the ribose moiety, or a site on the base through a linker group and the second tag comprises a molecular fluorescent tag covalently bonded to the α-phosphate, the γ-phosphate or the terminal phosphate through a linker group.