Mutant polymerases
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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.
144 Citations
82 Claims
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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.
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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)
- View Dependent Claims (72, 73, 74, 75, 76, 77)
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78. A method for preparing tagged polymerizing agents comprising the steps of:
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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. - View Dependent Claims (82)
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79. A method for preparing tagged polymerizing agents comprising the steps of:
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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.
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80. A method for preparing tagged polymerizing agents comprising the steps of:
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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.
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81. A method for preparing tagged polymerizing agents comprising the steps of:
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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.
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Specification