Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase.

Abstract:

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20-50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq DNA pol/TBD is thermostable, PCR competent and able to copy repetitive deoxynucleotide sequences six to seven times more faithfully than Taq DNA polymerase and makes 2-3-fold fewer AT-->GC transition mutations.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Fidelity, Nucleotide Incorporation, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Taq (exo-) Davidson JF2003 5-3' Exonuclease No
Taq (exo-) Davidson JF2003 Processivity 25bp
Taq (exo-) Davidson JF2003 Specific Activity 1630 units/mg Technique: Polymerase Assay (M13 DNA) (With thioredoxin)
Taq (exo-) Davidson JF2003 Specific Activity 1308 units/mg Technique: Polymerase Assay (M13 DNA) (Without thioredoxin)
TaqTBD (exo-) Davidson JF2003 Processivity 10bp
TaqTBD (exo-) Davidson JF2003 Specific Activity 6521 units/mg Technique: Polymerase Assay (M13 DNA) (With thioredoxin)
TaqTBD (exo-) Davidson JF2003 Specific Activity 201 units/mg Technique: Polymerase Assay (M13 DNA) (Without thioredoxin)
Taq pol I Davidson JF2003 5-3' Exonuclease Yes
Taq pol I Davidson JF2003 Nucleotide Substitution Rate 0.00192 errors/bp
Taq pol I Davidson JF2003 Processivity 80kb
Taq pol I Davidson JF2003 Specific Activity 2224 units/mg Technique: Polymerase Assay (M13 DNA) (With thioredoxin)
Taq pol I Davidson JF2003 Specific Activity 2218 units/mg Technique: Polymerase Assay (M13 DNA) (Without thioredoxin)
TaqTBD Davidson JF2003 5-3' Exonuclease Yes
TaqTBD Davidson JF2003 Nucleotide Substitution Rate 0.00148 errors/bp
TaqTBD Davidson JF2003 Processivity 300bp
TaqTBD Davidson JF2003 Specific Activity 1291 units/mg Technique: Polymerase Assay (M13 DNA) (With thioredoxin)
TaqTBD Davidson JF2003 Specific Activity 302 units/mg Technique: Polymerase Assay (M13 DNA) (Without thioredoxin)

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