Mutant polymerases

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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         27 . A mutant Taq polymerase comprising native Taq polymerase including a cysteine residue replacement at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661 and mixtures or combinations thereof.  
     
     
         28 . The polymerase of  claim 27 , further comprising a tag covalently bonded to the replacement cysteine site or sites through its or their SH group or groups.  
     
     
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         35 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 513 amino acid site of the native Taq polymerase.  
     
     
         36 . The polymerase of  claim 35 , wherein the replacement cysteine at the 513 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         37 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 514 amino acid site of the native Taq polymerase.  
     
     
         38 . The polymerase of  claim 37 , wherein the replacement cysteine at the 514 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         39 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 515 amino acid site of the native Taq polymerase.  
     
     
         40 . The polymerase of  claim 39 , wherein the replacement cysteine at the 515 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         41 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 516 amino acid site of the native Taq polymerase.  
     
     
         42 . The polymerase of  claim 41 , wherein the replacement cysteine at the 516 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         43 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 517 amino acid site of the native Taq polymerase.  
     
     
         44 . The polymerase of  claim 43 , wherein the replacement cysteine at the 517 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         45 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 518 amino acid site of the native Taq polymerase.  
     
     
         46 . The polymerase of  claim 45 , wherein the replacement cysteine at the 518 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         47 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 643 amino acid site of the native Taq polymerase.  
     
     
         48 . The polymerase of  claim 47 , wherein the replacement cysteine at the 643 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         49 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 647 amino acid site of the native Taq polymerase.  
     
     
         50 . The polymerase of  claim 49 , wherein the replacement cysteine at the 647 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         51 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 649 amino acid site of the native Taq polymerase.  
     
     
         52 . The polymerase of  claim 51 , wherein the replacement cysteine at the 649 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         53 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 653 amino acid site of the native Taq polymerase.  
     
     
         54 . The polymerase of  claim 53 , wherein the replacement cysteine at the 653 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         55 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 654 amino acid site of the native Taq polymerase.  
     
     
         56 . The polymerase of  claim 55 , wherein the replacement cysteine at the 654 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         57 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 655 amino acid site of the native Taq polymerase.  
     
     
         58 . The polymerase of  claim 57 , wherein the replacement cysteine at the 655 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         59 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 656 amino acid site of the native Taq polymerase.  
     
     
         60 . The polymerase of  claim 59 , wherein the replacement cysteine at the 656 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         61 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 657 amino acid site of the native Taq polymerase.  
     
     
         62 . The polymerase of  claim 61 , wherein the replacement cysteine at the 657 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         63 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 658 amino acid site of the native Taq polymerase.  
     
     
         64 . The polymerase of  claim 63 , wherein the replacement cysteine at the 658 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         65 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 659 amino acid site of the native Taq polymerase.  
     
     
         66 . The polymerase of  claim 65 , wherein the replacement cysteine at the 659 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         67 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 660 amino acid site of the native Taq polymerase.  
     
     
         68 . The polymerase of  claim 67 , wherein the replacement cysteine at the 660 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         69 . The polymerase of  claim 27 , wherein the native Taq polymerase includes a cysteine residue replacement at a 661 amino acid site of the native Taq polymerase.  
     
     
         70 . The polymerase of  claim 69 , wherein the replacement cysteine at the 661 site includes a tag covalently bonded to the cysteine through its SH group.  
     
     
         71 . A genetically engineered polymerizing agent comprising a native polymerizing agent including one cysteine residue replacement or a plurality of cysteine residue replacements at one site or a plurality of sites that (1) are not in contact with other proteins, (2) do not alter the conformation or folding of the polymerizing agent; and (3) are not involved in the function of the polymerizing agent. (I do not believe we have any support for a fusion pols)  
     
     
         72 . The agent of  claim 71 , wherein the site or sites surround active regions of the polymerizing agent that undergo changes during monomer incorporation.  
     
     
         73 . The agent of  claim 72 , wherein the regions are selected from the group consisting of the monomer binding regions, monomer incorporation regions, and pyrophosphate release regions. (Taken directly from text in the application!)  
     
     
         74 . The agent of  claim 71 , wherein the site is an internal site or sites are internal sites that reduce background signals by reducing interactions between the agent and non-specifically associated tagged monomers.  
     
     
         75 . The agent of  claim 71 , wherein the site or sites comprise sites in the agent that undergo the largest change in position during a monomer incorporation cycle.  
     
     
         76 . The agent of  claim 71 , wherein the polymerizing agent is a polymerase or a reverse transcriptase.  
     
     
         77 . The agent of  claim 76 , 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.  
     
     
         78 . A method for preparing tagged polymerizing agents comprising the steps of: 
 selecting one amino acid site or a plurality of amino acid sites of a polymerizing agent, where the site or sites (1) are not in contact with other proteins, (2) do not alter the conformation or folding of the polymerizing agent; and (3) are not involved in the function of the polymerizing agent;    preparing an DNA encoding sequence for the polymerizing agent replacing the amino acid codon at the site or sites with cysteine codons,    expressing the DNA encoding sequence in a microorganism to form a polymerizing agent variant including cysteines at the site or sites,    isolating the polymerizing agent variant, and    modifying the polymerizing agent variant with at least one tag adapted to react with the cysteine SH groups to form a tagged polymerizing agent,    where the tagged polymerizing agent is adapted for use in nucleic acid sequencing reactions.    
     
     
         79 . A method for preparing tagged polymerizing agents comprising the steps of: 
 selecting one amino acid site of a polymerizing agent, where the site (1) is not in contact with other proteins, (2) does not alter the conformation or folding of the polymerizing agent; and (3) is not involved in the function of the polymerizing agent;    preparing an DNA encoding sequence for the polymerizing agent replacing the amino acid codon at the site with a cysteine codon,    expressing the DNA encoding sequence in a microorganism to form a polymerizing agent variant including a cysteine at the site,    isolating the polymerizing agent variant, and    modifying the polymerizing agent variant with at least one tag adapted to react with the cysteine SH group to form a tagged polymerizing agent,    where the tagged polymerizing agent is adapted for use in nucleic acid sequencing reactions.    
     
     
         80 . A method for preparing tagged polymerizing agents comprising the steps of: 
 selecting two amino acid sites of a polymerizing agent, where the sites (1) are not in contact with other proteins, (2) do not alter the conformation or folding of the polymerizing agent; and (3) are not involved in the function of the polymerizing agent;    preparing an DNA encoding sequence for the polymerizing agent replacing the amino acid codon at the sites with cysteine codons,    expressing the DNA encoding sequence in a microorganism to form a polymerizing agent variant including cysteines at the sites,    isolating the polymerizing agent variant, and    modifying the polymerizing agent variant with a first tag and second tag, where the tags are adapted to react with the cysteine SH groups to form a tagged polymerizing agent,    where the tagged polymerizing agent is adapted for use in nucleic acid sequencing reactions.    
     
     
         81 . A method for preparing tagged polymerizing agents comprising the steps of: 
 selecting a target amino acid site from a plurality of amino acid sites of a polymerizing agent, where the sites (1) are not in contact with other proteins, (2) do not alter the conformation or folding of the polymerizing agent; and (3) are not involved in the function of the polymerizing agent;    modifying the target site with a tag under conditions where the other sites are protected or competitively inhibited, and    deprotecting the other sites,    where the tagged polymerizing agent is adapted for use in nucleic acid sequencing reactions.    
     
     
         82 . The agent of  claim 78 , wherein the sites were selected using a program to determine a change in position of amino acids in an open and a closed conformation of the polymerizing agent relative to a gamma phosphate of a bound ddGTP from two different crystal structures of the polymerizing agent containing the primer and bound ddGTP, where the selected amino acid sites undergo the largest change in position and where distances were calculated for each amino acid between their alpha and beta carbon atoms and the gamma phosphate group of the bound ddGTP.