Compositions for sequence determination using tagged polymerizing agents and tagged monomers

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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         10 . A composition comprising a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent, a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex, a collisional quencher, and monomers for the polymerizing agent, where one monomer type includes a first monomer tag, where at least one of the tags has a fluorescent property that undergoes a change before, during and/or after a sequence of monomer incorporations due to an interaction between the polymerizing agent tag and the monomer tag and where the collisional quencher is adapted to quench fluorescence from the tagged monomers in the medium.  
     
     
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         14 . The composition of  claim 10 , wherein the polymerizing agent is a polymerase or reverse transcriptase.  
     
     
         15 . The composition of  claim 14 , 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 and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.  
     
     
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         17 . The composition of  claim 10 , wherein the monomers comprise dNTPs and the monomer tags are covalently bonded to an β, γ or terminal phosphate group of the tagged monomer types.  
     
     
         18 . The composition of  claim 10 , wherein the fluorescent property comprises a duration, an intensity and/or frequency of emitted light.  
     
     
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         20 . The composition of  claim 15 , wherein the polymerase comprises Taq DNA polymerase I having the fluorescent tag attached at an amino acid site of the Taq DNA polymerase I selected from the group consisting of 513-518, 643, 647, 649 and 653-661.  
     
     
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         35 . The composition of  claim 10 , wherein each monomer type includes a fluorescent tag, and each fluorescent tag is the same or different.  
     
     
         36 . The composition of  claim 10 , wherein at least two monomer types include a fluorescent tag, and each fluorescent tag is the same or different.  
     
     
         37 . The composition of  claim 10 , wherein at least three monomer types include a fluorescent tag, and each fluorescent tag is the same or different.  
     
     
         38 . A composition comprising: 
 a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,    a template,    a primer,    nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the β, γ and/or terminal phosphate group thereof, and    a collisional quencher adapted to quench the fluorescence of the t agged d NTPs in the medium,    where at least one ofthe fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.    
     
     
         39 . The composition of  claim 38 , wherein the polymerizing agent is a polymerase or reverse transcriptase.  
     
     
         40 . The composition of  claim 39 , 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 and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.  
     
     
         41 . The composition of  claim 40 , wherein the polymerase comprises Taq DNA polymerase I having a tag attached to an amino acid at a specific amino acid position of the Taq DNA polymerase I, where the amino acid position is selected from the group consisting of 513-518, 643, 647, 649 and 653-661 of SEQ. ID No. 11.  
     
     
         42 . The composition of  claim 38 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.  
     
     
         43 . The composition of  claim 38 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.  
     
     
         44 . The composition of  claim 43 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         45 . The composition o f  claim 43 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         46 . The composition of  claim 43 , wherein each dNTP type includes a fluorescent tag, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         47 . A composition comprising: 
 a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,    a template,    a primer,    nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the β terminal phosphate group thereof, and    a collisional quencher a dapted to quench the fluorescence of the tagged dNTPs in the medium,    where at least one ofthe fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.    
     
     
         48 . The composition of  claim 47 , wherein the polymerizing agent is a polymerase or reverse transcriptase.  
     
     
         49 . The composition of  claim 47 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.  
     
     
         50 . The composition of  claim 47 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.  
     
     
         51 . The composition of  claim 50 , wherein at least two DNTP types include fluorescent tags, where the tags for each dNTP type are the same or different and each dNTP tag form a FRET pair with the polymerizing agent tag.  
     
     
         52 . The composition of  claim 50 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         53 . The composition of  claim 50 , wherein each dNTP type includes a fluorescent tag, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         54 . A composition comprising: 
 a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the polymerizing agent,    a template,    a primer,    nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the γ phosphate group thereof, and    a collisional quencher adapted to quench the fluorescence of the tagged dNTPs in the medium,    where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.    
     
     
         55 . The composition of  claim 54 , wherein the polymerizing agent is a polymerase or reverse transcriptase.  
     
     
         56 . The composition of  claim 54 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.  
     
     
         57 . The composition of  claim 54 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.  
     
     
         58 . The composition of claim 57 , wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         59 . The composition of  claim 57 , wherein at least three dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         60 . The composition of  claim 57 , wherein each dNTP type includes a fluorescent tag, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         61 . A composition comprising: 
 a polymerizing agent including a polymerizing agent fluorescent tag at or near the polymerizing agent, associated with the polymerizing agent or covalently bonded to a site on the    polymerizing agent,    a template,    a primer,    nucleotide triphosphates (dNTPs) for the polymerizing agent, where one dNTP type includes a monomer fluorescent tag covalently bonded to the terminal phosphate group thereof, and    a collisional quencher adapted to quench the fluorescence of the tagged dNTPs in the medium,    where at least one of the fluorescent tags has a fluorescence property that undergoes a change before, during and/or after each of a sequence of tagged dNTP incorporations due to an interaction between the polymerizing agent tag and the dNTP tag.    
     
     
         63 . The composition of  claim 61 , wherein the polymerizing agent is a polymerase or reverse transcriptase.  
     
     
         64 . The composition of  claim 61 , wherein the fluorescence property comprises a duration, an intensity and/or frequency of emitted fluorescent light.  
     
     
         65 . The composition of  claim 61 , wherein the dNTP tag and the polymerizing agent form a fluorescence resonance energy transfer (FRET) pair resulting in acceptor fluorescence, which occurs when the dNTP tag and the polymerizing agent tag are in close proximity before, during and/or after each tagged dNTP incorporation.  
     
     
         66 . The composition of claim 65, wherein at least two dNTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         67 . The c omposition of  claim 65 , wherein at least t hree d NTP types include fluorescent tags, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         68 . The composition of  claim 65 , wherein each dNTP type includes a fluorescent tag, where the tags for each dNTP type are the same or different, and where each dNTP tag forms a FRET pair with the polymerizing agent tag.  
     
     
         69 . The composition of claims  36 ,  38 ,  47 ,  54 , or  61 , wherein each fluorescent tag is bonded to its monomer type via a linker.