Mutant chimeric DNA polymerase
First Claim
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1. A thermostable DNA polymerase consisting of an N-terminal region and a C-terminal region,wherein said N-terminal region consists of amino acids 1 through n of a Thermus species DNA polymerase, wherein n is an amino acid corresponding to an amino acid m of a Thermatoga maritima (Tma) DNA polymerase described by SEQ ID NO:
- 10, wherein m is between 137 and 291;
wherein said C-terminal region consists of amino acids m+1 through 893 of said Tma DNA polymerase;
wherein said N-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in said Thermus species DNA polymerase, or said C-terminal region is modified by at least one point mutation within the region that is amino acids m+1 to 291 of Tma DNA polymerase, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in Tma DNA polymerase;
wherein said C-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces 3′
to 5′
exonuclease activity when present in Tma DNA polymerase; and
wherein said C-terminal region is modified to contain a tyrosine at amino acid 730.
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Abstract
Mutant, chimeric thermostable DNA polymerases are provided, along with purified DNA sequences that encode the enzymes. Also provided are methods for producing and using the enzymes.
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Citations
63 Claims
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1. A thermostable DNA polymerase consisting of an N-terminal region and a C-terminal region,
wherein said N-terminal region consists of amino acids 1 through n of a Thermus species DNA polymerase, wherein n is an amino acid corresponding to an amino acid m of a Thermatoga maritima (Tma) DNA polymerase described by SEQ ID NO: - 10, wherein m is between 137 and 291;
wherein said C-terminal region consists of amino acids m+1 through 893 of said Tma DNA polymerase;
wherein said N-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in said Thermus species DNA polymerase, or said C-terminal region is modified by at least one point mutation within the region that is amino acids m+1 to 291 of Tma DNA polymerase, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in Tma DNA polymerase;
wherein said C-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces 3′
to 5′
exonuclease activity when present in Tma DNA polymerase; and
wherein said C-terminal region is modified to contain a tyrosine at amino acid 730. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 55, 56, 57, 58, 59, 60, 61, 62, 63)
- 10, wherein m is between 137 and 291;
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10. An isolated DNA that encodes a thermostable DNA polymerase, wherein said thermostable DNA polymerase consists of an N-terminal region and a C-terminal region,
wherein said N-terminal region consists of amino acids 1 through n of a Thermus species DNA polymerase, wherein n is an amino acid corresponding to an amino acid m of a Thermatoga maritima (Tma) DNA polymerase described by SEQ ID NO: - 10, wherein m is between 137 and 291;
wherein said C-terminal region consists of amino acids m+1 through 893 of said Tma DNA polymerase;
wherein said N-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in said Thermus species DNA polymerase, or said C-terminal region is modified by at least one point mutation within the region that is amino acids m+1 to 291 of Tma DNA polymerase, each characterized in that it substantially reduces or eliminates 5′
-nuclease activity when present in Tma DNA polymerase;
wherein said C-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces 3′
to 5′
exonuclease activity when present in Tma DNA polymerase; and
wherein said C-terminal region is modified to contain a tyrosine at amino acid 730. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
(a) culturing a host cell of claim 37 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
- 10, wherein m is between 137 and 291;
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47. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 38 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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48. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 39 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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49. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 40 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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50. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 41 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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51. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 42 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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52. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 43 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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53. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 44 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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54. A method for preparing a thermostable DNA polymerase, comprising:
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(a) culturing a host cell of claim 45 under conditions which promote the expression of thermostable DNA polymerase; and
(b) isolating thermostable DNA polymerase from said host cell.
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Specification