US5489523A
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Exonuclease-deficient thermostable Pyrococcus furiosus DNA polymerase I
Assignee: Agilent Technologies Inc
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Filing: 1992-10-26
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Granted: 1996-02-06
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Expires: 2012-10-26
Abstract
Abstract A recombinant thermostable Pyrococcus furiosus DNA polymerase is described that is deficient in 3'-5' exonuclease activity that is useful in DNA cycle sequencing reactions.
Inventors
- 👤 Eric J. Mathur
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 13:41
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12Y; C12Y207/00; C12Y207/07; C12Y207/07007
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
74 words
A recombinant thermostable Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 2
Dependent Claim
74 words
A recombinant thermostable Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 3
Dependent Claim
25 words
The polymerase of claim 1 wherein said polymerase retains at least 95% of its DNA polymerase activity after exposure to 95° C. for 1 hour.
Claim 4
Dependent Claim
24 words
The polymerase of claim 1 wherein said polymerase has less than 50 units of specific 3' to 5' exonuclease activity per milligram of protein.
Claim 5
Dependent Claim
28 words
The polymerase of claim 1 wherein said polymerase has a ratio of specific activity of DNA polymerase activity to 3' to 5' exonuclease activity of at least 1000.
Claim 6
Dependent Claim
81 words
A plasmid containing a gene coding for a recombinant thermostable Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 7
Dependent Claim
81 words
A plasmid containing a gene coding for a recombinant thermostable Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 8
Dependent Claim
28 words
The plasmid of claim 5 wherein said polymerase has a ratio of specific activity of DNA polymerase activity to 3' to 5' exonuclease activity of at least 1000.
Claim 9
Dependent Claim
10 words
A procaryotic cell transformed with the plasmid of claim 5.
Claim 10
Dependent Claim
79 words
A bacteriophage containing a gene coding for a Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 11
Dependent Claim
79 words
A bacteriophage containing a gene coding for a Pyrococcus furiosus DNA polymerase I deficient in 3' to 5' exonuclease activity, wherein said polymerase has an amino acid residue sequence represented by the formula shown in SEQ ID NO 1 from residue 1 to residue 775, with the exception of an amino acid residue substitution selected from the group consisting of (1) Ala 141 /Ala 143 , (2) Ala 214 /Ala 215 , and (3) Ala 311 /Ala 315 .
Claim 12
Dependent Claim
28 words
The bacteriophage of claim 8 wherein said polymerase has a ratio of specific activity of DNA polymerase activity to 3' to 5' exonuclease activity of at least 100.
References Cited (Backward Citations)
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