High fidelity DNA polymerase compositions and uses therefor
AssigneeAgilent Technologies, Inc.
InventorsHolly Hogrefe, Michael Borns, Joseph A. Sorge
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Abstract
The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 3′-5′ exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.
Claims74
An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme comprises a DNA polymerization activity, and said second enzyme comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is a wild type Pfu DNA polymerase, said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme is a Taq DNA polymerase, said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme comprises a DNA polymerization activity and is a wild-type Pfu DNA polymerase or a wild-type Taq DNA polymerase, and said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
An enzyme mixture comprising two or more enzymes, wherein at least a first enzyme in said enzyme mixture comprises a DNA polymerization activity, and at least a second enzyme in said enzyme mixture comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A mutant Pfu DNA polymerase with reduced DNA polymerization activity, wherein said mutant Pfu DNA polymerase comprises one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
A composition comprising a mutant Pfu DNA polymerase, wherein said mutant DNA polymerase comprises one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
A mutant Pfu DNA polymerase produced by introducing a mutation in to a polynucleotide encoding a wild type Pfu DNA polymerase to produce a mutant Pfu DNA polymerase comprising one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
A mutant Pfu DNA polymerase comprising a reduced DNA polymerization activity, wherein said mutant Pfu DNA polymerase is produced by the steps:
- (a) providing a polynucleotide encoding a wild-type Pfu DNA polymerase;
- (b) introducing one or more nucleotide mutations into said polynucleotide to produce a mutant polynucleotide encoding said mutant Pfu DNA polymerase;
- and
- (c) expressing said mutant polynucleotide to produce said mutant Pfu DNA polymerase, wherein said mutant Pfu DNA polymerase comprises one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
A composition comprising a mutant Pfu DNA polymerase produced by expressing a polynucleotide encoding a Pfu DNA polymerase with a reduced DNA polymerization activity, wherein said mutant Pfu DNA polymerase comprises one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
A composition comprising a mutant Pfu DNA polymerase comprising a reduced DNA polymerization activity, wherein said mutant Pfu DNA polymerase is produced by the steps:
- (a) introducing a mutation into a polynucleotide encoding a wild-type Pfu DNA polymerase to produce a mutant polynucleotide encoding said mutant Pfu DNA polymerase comprising one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388;
- (b) expressing said mutant polynucleotide to produce said composition comprising said mutant Pfu DNA polymerase.
A kit comprising a first enzyme and a second enzyme, wherein said first enzyme comprises a DNA polymerization activity, said second enzyme comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity, and packaging material therefore.
A kit comprising a first enzyme and a second enzyme, and packaging material therefor, wherein said first enzyme is a wild type Pfu DNA polymerase, said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A kit comprising a first enzyme and a second enzyme, and packaging material therefore, wherein said first enzyme is a Taq DNA polymerase, and packaging material therefor, said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A kit comprising an enzyme mixture which comprises a first enzyme and a second enzyme, wherein said first enzyme comprises a DNA polymerization activity and is a wild-type Pfu DNA polymerase or a wild-type Taq DNA polymerase, and said second enzyme is a mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity, and packaging means therefor.
A kit comprising a mutant DNA polymerase which comprises a reduced DNA polymerization activity and packaging material therefor, wherein said mutant Pfu DNA polymerase comprises one or more mutations at amino acid positions selected from the group consisting of:
- T542, D543, K593, Y595, Y385, G387, and G388.
An isolated polynucleotide comprising a nucleotide sequence encoding a mutant enzyme comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A pair of isolated polynucleotides, wherein a first polynucleotide of said pair comprises a polynucleotide sequence encoding a first enzyme comprising a DNA polymerase activity, and a second polynucleotide of said pair comprises a polynucleotide sequence encoding an enzyme comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A pair of isolated polynucleotides, wherein a first polynucleotide of said pair comprises a polynucleotide sequence encoding a wild-type Pfu DNA polymerase or a Taq DNA polymerase, and a second polynucleotide of said pair comprises a polynucleotide sequence encoding an mutant Pfu DNA polymerase comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity.
A method for DNA synthesis comprising:
- (a) providing an enzyme mixture, said enzyme mixture comprising a first enzyme comprising a DNA polymerization activity, and a second enzyme comprising a 3′-5′ exonuclease activity and a reduced DNA polymerization activity;
- and
- (b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis.
A method for DNA synthesis comprising:
- (a) providing an enzyme mixture, said enzyme mixture comprising a wild type Pfu DNA polymerase as a first enzyme, and a mutant Pfu DNA polymerase as a second enzyme which comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity;
- and
- (b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis.
A method for TA cloning of DNA synthesis product comprising:
- (a) providing an enzyme mixture, said enzyme mixture comprising a Taq DNA polymerase as a first enzyme, and a mutant Pfu DNA polymerase as a second enzyme which comprises a 3′-5′ exonuclease activity and a reduced DNA polymerization activity;
- (b) contacting said enzyme mixture with a nucleic acid template, wherein said enzyme mixture permits DNA synthesis to generate a synthesized DNA product;
- and
- (c) inserting said synthesized DNA product into a TA cloning vector.
Dependencies are inferred from the claim text.