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Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR
M Takagi1, M Nishioka, H Kakihara
1Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Applied and Environmental Microbiology
|November 15, 1997
Summary
The KOD DNA polymerase from Pyrococcus sp. strain KOD1 offers enhanced performance for polymerase chain reaction (PCR). This archaeal enzyme exhibits a significantly faster extension rate and higher processivity than Pfu DNA polymerase.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The DNA polymerase gene from Pyrococcus sp. strain KOD1 (KOD DNA polymerase) was analyzed.
- It contains a 5,013-base open reading frame encoding 1,671 amino acids.
- The gene includes two intervening sequences (inteins) within conserved regions of alpha-like DNA polymerases.
Purpose of the Study:
- To express and characterize the mature KOD DNA polymerase.
- To confirm its enzymatic activities and compare its performance to other DNA polymerases.
Main Methods:
- Gene expression in Escherichia coli.
- Purification and characterization of the recombinant enzyme.
- Enzymatic assays to determine activity, optimal temperature, mutation frequency, extension rate, and processivity.
Main Results:
- The KOD DNA polymerase possesses 3'-5' exonuclease activity.
- Its optimal temperature (75°C) and mutation frequency (3.5 x 10^-3) are comparable to Pfu DNA polymerase.
- KOD DNA polymerase shows a 5-fold higher extension rate (100-130 nucleotides/s) and 10-15 times greater processivity.
Conclusions:
- KOD DNA polymerase is a highly efficient enzyme for DNA amplification.
- Its superior extension rate and processivity enable more accurate PCR with reduced reaction times.