Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis.

Abstract:

DNA synthesis fidelities of two thermostable DNA polymerases, Thermus aquaticus (Taq) and Thermococcus litoralis (Tli, also known as Vent), and a non-thermostable enzyme, a modified T7 DNA polymerase (Sequenase), were determined by analyzing polymerase chain reaction (PCR) products using denaturing gradient gel electrophoresis (DGGE). The error rates were 4.4, 8.9, and 2.4 x 10(-5) errors/bp for modified T7, Taq, and Tli polymerase, respectively. Reducing the nucleotide triphosphate concentration for Tli polymerase during PCR did not alter the fidelity. The ability of DGGE to detect a mutant present at several percent in a wild type population is related to the polymerase fidelity. To examine the sensitivity of mutant detection, human genomic DNA containing a 1% fraction of a known base pair substitution mutant was PCR-amplified with the three enzymes using primers that flank the mutant sequence. The PCR products were analyzed by DGGE. The signal from the mutant present at 1% was visible in the samples amplified with modified T7 and Tli polymerase, but the higher error rate of Taq polymerase did not permit visualization of the signal in DNA amplified with Taq polymerase.

Polymerases:

Topics:

Biotech Applications, Fidelity, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Taq pol I Cariello NF1991 3-5' Exonuclease (proofreading) No
Taq pol I Cariello NF1991 Nucleotide Substitution Rate 8.9E-05 errors/bp
Taq pol I Cariello NF1991 Application name PCR
Vent Cariello NF1991 Application name PCR
Sequenase Cariello NF1991 3-5' Exonuclease (proofreading) No
Sequenase Cariello NF1991 Nucleotide Substitution Rate 4.4E-05 errors/bp Technique: DGGE (2mM MgSO4)
Sequenase Cariello NF1991 Application name PCR
Vent Cariello NF1991 3-5' Exonuclease (proofreading) Yes
Vent Cariello NF1991 Nucleotide Substitution Rate 2.4E-05 errors/bp Technique: DGGE (2mM MgSO4)

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