DNA polymerase having modified nucleotide binding site for DNA sequencing
First Claim
1. Modified gene encoding a modified Pol I-type DNA polymerase wherein said modified gene is modified to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said modified DNA polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to the ability of a corresponding naturally-occurring unmodified DNA polymerase by at least 20-fold.
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Accused Products
Abstract
Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.
240 Citations
108 Claims
- 1. Modified gene encoding a modified Pol I-type DNA polymerase wherein said modified gene is modified to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said modified DNA polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to the ability of a corresponding naturally-occurring unmodified DNA polymerase by at least 20-fold.
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11. Method for production of a modified Pol I-type DNA polymerase having an increased ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase comprising steps of:
- providing a nucleic acid molecule encoding a DNA polymerase and mutagenizing said nucleic acid molecule to incorporate one or more base changes in nucleotide base sequence at a region that encodes its dNMP binding site to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in the dNMP binding site to alter ability of said polymerase encoded by said nucleic acid to incorporate a dideoxynucleotide by at least 20-fold.
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12. Method for determining a nucleotide base sequence of a DNA molecule comprising the steps of:
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incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleoside triphosphate, a Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site compared to a naturally-occurring unmodified DNA polymerase to have at least a 20-fold increased ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to said naturally-occurring unmodified polymerase, said polymerase having sufficient DNA polymerase activity and less than 1000 units exonuclease activity per mg DNA polymerases, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, in an incubating reaction; and separating the DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined. - View Dependent Claims (13, 14, 15, 16, 17, 18)
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19. A kit for DNA sequencing comprising a modified Pol I-type DNA polymerase modified to include a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site of the DNA polymerase to increase the ability of said DNA polymerase to incorporate a dideoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase by at least 20-fold relative to a corresponding deoxynucleotide;
- and a reagent necessary for said sequencing selected from the group consisting of dITP, a chain terminating agent, deaza-GTP and a manganese-containing compound.
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20. A method for sequencing a strand of DNA comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said strand, to form an hybridized mixture, incubating said hybridized mixture with one or more deoxyribonucleoside triphosphates, a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide at least 20-fold relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each said second DNA product having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
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21. Method for sequencing a nucleic acid comprising:
combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide at least 20-fold relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, and at least two chain terminating agents in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced;
separating the nucleic acid fragments by size; and
determining nucleic acid sequence wherein said agents are differentiated from each other by intensity of a label in the primer extension products.
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22. A method for in vitro mutagenesis of a cloned DNA fragment comprising providing said cloned fragment and a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide at least 20-fold relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, contacting said cloned fragment with said polymerase under conditions for synthesizing a DNA strand from said fragment, wherein said conditions cause formation of said DNA strand by incorporation of a plurality of individual contiguous bases able to base-pair with said fragment and incorporation of a nucleotide base unable to base pair with said fragment.
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23. A method for in vitro mutagenesis of a template DNA fragment comprising providing a primer and said template, said primer having contiguous bases able to base-pair with contiguous bases of said template except at least one base which is unable to base-pair with said template, and extending said primer with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase at least 20-fold relative to a corresponding deoxynucleotide to incorporate a dideoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase.
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24. A method for producing blunt-ended double-stranded DNA from a linear DNA molecule having a 5'"'"' end comprising a single-stranded region, wherein 3'"'"' end of said molecule is double stranded and has no 3'"'"' protruding termini, comprising incubating said DNA molecule with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase at least 20-fold to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, wherein said polymerase acts on said single-stranded region to produce a blunt-ended double stranded DNA molecule.
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25. A method for labeling a 3'"'"' end of a DNA fragment comprising incubating said DNA fragment with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase at least 20-fold to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, and a labelled deoxynucleotide species under conditions in which said 3'"'"' end of said DNA fragment is extended by said polymerase and thereby labelled by addition of said labelled deoxynucleotide to said DNA fragment.
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26. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double-stranded DNA sequence to form an annealed mixture and incubating the annealed mixture with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability at least 20-fold of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence with their 3'"'"' ends directed towards each other after annealing, and with the DNA sequence to be amplified located between the first and second annealed primers.
- 27. A Thermus aquaticus DNA polymerase having a tyrosine at residue 667.
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28. An E. coli DNA polymerase I having a tyrosine at residue 762.
- 29. Purified Pol I type DNA polymerase having a tyrosine residue at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site provided that said polymerase is not a T7-type DNA polymerase or a mitochondrial DNA polymerase.
- 31. A modified alpha-type DNA polymerase comprising the sequence K N1 N2 N3 N4 N5 N6 Y G, at its dNMP binding site wherein each N is independently any amino acid, and wherein one said N is mutated to have a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to provide a polymerase which does not discriminate against ddNMP incorporation more than 50 fold relative to the corresponding dNMP incorporation.
- 33. Recombinant DNA polymerase which in the presence of magnesium as the only added divalent cation has an average processivity of less than 100 and discriminates less than 100 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide, wherein said DNA polymerase is not reverse transcriptase and wherein said DNA polymerase includes a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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34. Recombinant DNA polymerase which in the presence of magnesium as the only divalent cation has an average processivity of less than 50 and discriminates less than 50 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide and wherein said DNA polymerase includes a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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35. Recombinant DNA polymerase which in the presence of magnesium as the only divalent cation has an average processivity of less than 50 and discriminates less than 5 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide and wherein said DNA polymerase includes a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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36. Recombinant Thermophilic DNA polymerase that discriminates against a ddNMP relative to a corresponding deoxynucleotide by less than a factor of 100 and wherein said DNA polymerase includes a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
- 37. Recombinant Thermophilic DNA polymerase that discriminates in the presence of magnesium as the only divalent cation against a ddNMP relative to a corresponding deoxynucleotide by less than a factor of 100 and wherein said DNA polymerase includes a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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42. A purified cellular DNA polymerase having a tyrosine residue in place of the naturally occurring amino acid residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site which causes said polymerase not to discriminate against a ddNMP relative to a corresponding deoxynucleotide by more than 50 fold.
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44. Method for cycle sequencing, comprising step of providing an excess or equal amount of all four dNTPs compared to each of the four ddNTPs in a cycle sequencing reaction and performing said cycle sequencing reaction using a DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNTP binding site that does not discriminate against incorporation of a ddNMP relative to a corresponding deoxynucleotide by more than 50-fold.
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45. Method for cycle sequencing comprising step of providing less than 10 times of an amount of all four fluorescently labelled dideoxynucleotide than a corresponding deoxynucleotides in a cycle sequencing reaction and performing said cycle sequencing reaction using a DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site that does not discriminate against incorporation of a ddNMP relative to a corresponding deoxynucleotide by more than 50-fold.
- 46. A purified thermostable DNA polymerase having a deoxynucleotide binding site with the sequence K N1 N2 N3 N4 N5 N6 N7 Y G/Q wherein each N1 -N3 and N5 -N7 is independently any amino acid and N4 is a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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63. Method for production of a modified DNA polymerase comprising steps of:
- providing a nucleic acid molecule encoding a thermostable DNA polymerase comprising the sequence K N1 N2 N3 N4 N5 N6 N7 Y G/Q at its dNMP binding site wherein each N is independently any amino acid and mutagenizing said nucleic acid molecule to incorporate one or more base changes in the nucleotide base sequence to encode a tyrosine residue at position N4 corresponding to T7 DNA polymerase residue 526 or at to E. coli DNA polymerase residue 762 in its dNMP binding site.
- 64. A purified Pol I-type DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site, whereby ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide is increased 20-fold compared to ability of a corresponding naturally occurring unmodified DNA polymerase.
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70. A method for production of a modified DNA polymerase having an increased ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally occurring unmodified DNA polymerase which comprises the steps of modifying a nucleic acid molecule encoding a DNA polymerase to incorporate one or more base changes in its nucleotide base sequence to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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71. A method for determining the nucleotide base sequence of a DNA molecule comprising the steps of:
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incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleoside triphosphate, a DNA polymerase which in the presence of magnesium as the only added divalent cation discriminates less than 100 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide provided that said DNA polymerase is not reverse transcriptase, or a T7-type DNA polymerase, wherein said polymerase has a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 525 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 at its dNMP binding site, said polymerase having sufficient DNA polymerase activity to be useful for DNA sequencing and less than 1000 units exonuclease activity per mg DNA polymerase, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, in an incubating reaction; and separating the DNA products of the incubating reaction according to the size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determinated.
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72. A method for sequencing a strand of DNA comprising steps of:
- providing said strand hybridized with a primer able to hybridize to said strand, to form a hybridized mixture, incubating said hybridized mixture with one or more deoxyribonucleoside triphosphates, a DNA polymerase which is the presence of magnesium as the only added divalent cation discriminates less than 100 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide provided that said DNA polymerase is not reverse transcriptase, or a T7-type DNA polymerase, wherein said polymerase has a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 525 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in length of the elongated primer, each of said first DNA products having said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in length of the elongated primer, each said second DNA product having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by 1 to 20 bases, and being distinctively different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
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73. Method for sequencing a nucleic acid comprising:
combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a DNA polymerase which in the presence of magnesium as the only added divalent cation discriminates less than 100 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide provided that said DNA polymerase is not reverse transcriptase, or a T7-type DNA polymerase, and wherein said polymerase has a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 525 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.
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74. Method for determining a nucleotide base sequence of a DNA molecule comprising steps of incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleotide triphosphate, a DNA polymerase having a tyrosine residue at an amino acid position corresponding to E. coli DNA polymerase I residue 762, said polymerase having sufficient DNA polymerase activity for use in DNA sequencing and having less than 1000 units low exonuclease activity, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base in an incubating reaction;
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separating DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined, provided that the DNA polymerase is not a T7-type DNA polymerase. - View Dependent Claims (82)
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- 75. A kit for DNA sequencing comprising a DNA polymerase having a tyrosine residue at an amino acid position corresponding to E. coli DNA polymerase I residue 762, said polymerase having sufficient DNA polymerase activity for use in a DNA sequencing reaction and less than 1000 units per exonuclease activity per mg DNA polymerase, and a reagent necessary for said sequencing selected from the group consisting of dITP, a chain terminating agent and deaza-GTP provided that said polymerase is not a T7-type DNA polymerase.
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76. A kit for DNA sequencing comprising pyrophosphatase and a DNA polymerase which in the presence of magnesium as the only added divalent cation discriminates less than 100 times against incorporation of a ddNMP relative to a corresponding deoxynucleotide provided that said DNA polymerase has a tyrosine at an amino acid position corresponding to T7 DNA polymerase residue 525 in its dNMP binding site or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site, is not reverse transcriptase, or a T7-type DNA polymerase.
- 77. A kit for DNA sequencing comprising a DNA polymerase having a tyrosine residue at an amino acid position corresponding to E. coli DNA polymerase I residue 762 and a pyrophosphatase provided that the DNA polymerase is not a T7-type DNA polymerase.
- 83. Modified gene encoding a modified Pol II-type DNA polymerase wherein said modified gene is modified to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said modified DNA polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase.
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89. Method for production of a modified Pol II-type DNA polymerase having an increased ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase comprising steps of:
- providing a nucleic acid molecule encoding a DNA polymerase and mutagenizing said nucleic acid molecule to incorporate one or more base changes in nucleotide base sequence at a region that encodes its dNMP binding site to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase the ability of said polymerase encoded by said nucleic acid to incorporate a dideoxynucleotide.
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90. Method for determining the nucleotide base sequence of a DNA molecule comprising steps of:
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incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleoside triphosphate, a Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site compared to a naturally-occurring unmodified DNA polymerase to have increased ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to said naturally-occurring unmodified polymerase, said polymerase having sufficient DNA polymerase activity for use in DNA sequencing and less than 1000 units exonuclease activity per mg DNA polymerases, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base in an incubating reaction; and separating DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined. - View Dependent Claims (91, 92, 93, 94, 95, 96)
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97. A kit for DNA sequencing comprising a modified Pol II-type DNA polymerase modified to include a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said DNA polymerase to incorporate a dideoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase relative to a corresponding deoxynucleotide;
- and a reagent necessary for said sequencing selected from the group consisting of dITP, a chain terminating agent, deaza-GTP and a manganese-containing compound.
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98. A method for sequencing a strand of DNA comprising steps of:
- providing said strand hybridized with a primer able to hybridize to said strand, to form an hybridized mixture, incubating said hybridized mixture with one or more deoxyribonucleoside triphosphates, a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of the corresponding naturally-occurring unmodified DNA polymerase, and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in length of elongated primer, each said first DNA product having said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all first DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each of said second DNA products having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
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99. Method for sequencing a nucleic acid comprising:
combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to a ability of a corresponding naturally-occurring unmodified DNA polymerase, and at least two chain terminating agents in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced;
separating nucleic acid fragments by size; and
determining nucleic acid sequence wherein said agents are differentiated from each other by intensity of a label in primer extension products formed.
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100. A method for in vitro mutagenesis of a cloned DNA fragment comprising providing said cloned fragment and a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, contacting said cloned fragment with said polymerase under conditions for synthesizing a DNA strand from said fragment, wherein said conditions cause formation of said DNA strand by incorporation of a plurality of individual contiguous bases able to base-pair with said fragment and incorporation of a nucleotide base unable to base pair with said fragment.
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101. A method for in vitro mutagenesis of a template DNA fragment comprising providing a primer and said template, said primer having contiguous bases able to base-pair with contiguous bases of said template except at least one base which is unable to base-pair with said template, and extending said primer with a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase relative to a corresponding deoxynucleotide to incorporate a dideoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase.
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102. A method for producing blunt-ended double-stranded DNA from a linear DNA molecule having a 5'"'"' end comprising a single-stranded region, wherein the 3'"'"' end of said molecule is double stranded and has no 3'"'"' protruding termini, comprising incubating said DNA molecule with a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, wherein said polymerase acts on said single-stranded region to produce a blunt-ended double stranded DNA molecule.
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103. A method for labeling a 3'"'"' end of a DNA fragment comprising incubating said DNA fragment with a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase and a labelled deoxynucleotide species under conditions in which said 3'"'"' end of said DNA fragment is extended by said polymerase and thereby labelled by addition of said labelled deoxynucleotide to said DNA fragment.
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104. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double-stranded DNA sequence and incubating the annealed mixture with a modified Pol II-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site to increase ability of said polymerase to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide compared to ability of a corresponding naturally-occurring unmodified DNA polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence with their 3'"'"' ends directed towards each other after annealing, and with the DNA sequence to be amplified located between the first and second primers.
- 105. A purified Pol II-type DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site, whereby ability to incorporate a dideoxynucleotide relative to a corresponding deoxynucleotide is increased compared to ability of a corresponding naturally occurring unmodified DNA polymerase.
Specification