DESCRIPTION
KOD -Plus- is based on DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1(1)(2) KOD -Plus- exhibits excellent high PCR fidelity and efficiency. The enzyme solution of KOD -Plus- contains two types of anti-KOD DNA polymerase antibodies that inhibit polymerase and 3'→5' exonuclease activity, thus allowing for Hot Start PCR(3). KOD -Plus- generates blunt-end PCR products, due to 3'→5' exonuclease (proof-reading) activity.
FEATURES
- The PCR error ratio of KOD -Plus- is approximately 80 times less than that of Taq DNA polymerase.
- Effective for the amplification of GC-rich targets.
- Hotstart technology enables highly efficient amplification.
- KOD -Plus- enables the following amplifications (maximum): 21 kb from lambda phage DNA, 12 kb from human genomic DNA, and 7 kb from cDNA.
Amplification of a GC-rich target
- 1:KOD -Plus-
- 2:Taq DNA polymarase
- 3:Taq DNA polymarase + 5%DMSO
- 4:Taq DNA polymarase + PCR ehnancer
- 5:High efficent Taq DNA polymarase
- M:200bp Laddr Markers
Table 1.Comparison of the mutation frequency of each PCR enzyme.
| Colony number | Mutation frequency (%) |
||
|---|---|---|---|
| Total | Mutant | ||
| KOD -Plus- | 10,610 | 10 | 0.09 |
| High fidelity PCR enzyme (A company) | 10,900 | 68 | 0.62 |
| Pfu based DNA polymerase | 6,520 | 76 | 1.17 |
| Taq DNA polymerase | 10,560 | 780 | 7.39 |
*PCR fidelity was based on the mutation frequency of PCR products using a positive-selection base assay with the rpsL gene(4).
APPLICATIONS
High fidelity PCR
SOURCE
E. coli strain carrying the cloned KOD DNA polymerase gene
UNIT DEFNITION
One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTP into an acid insoluble material in 30 min at 75ºC.
STORAGE CONDITION
50 mM Tris-HCl (pH8.0), 0.1 mM EDTA, 1 mM DTT, 0.001% Tween 20, 0.001% Nonidet P-40, 50% Glycerol Store at -20ºC
COMPONENTS
This reagent includes the following components for 200 reactions:
| KOD -Plus-(1.0U/µL)* | 200 µL × 1 |
| 10× Buffer for KOD -Plus- | 1.0 µL × 1 |
| 25 mM MgSO4 | 1.0 µL × 1 |
| 2 mM dNTPs | 1.0 µL × 1 |
*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3'→5' exonuclease activity.
TYPICAL PCR REACTION SETUP
| Component | Volumes | Final Concentration |
|---|---|---|
| 10x Buffer for KOD -Plus- | 5 µL | 1× |
| 2mM dNTPs* | 5 µL | 0.2 µM each |
| 25mM MgSO4 | 2 µL | 1.0 mM |
| 10 pmol/µL Primer #1 | 1.5 µL | 0.3 µM |
| 10 pmol/µL Primer #2 | 1.5 µL | 0.3 µM |
| Template DNA | X µL | Genomic DNA 10~200 ng/50 µL Plasmid DNA 1~50 ng/50 µL cDNA ~100 ng (RNA equiv.)/50 µL |
| PCR grade water | Y µL | - |
| KOD-Plus- (1.0 U/µL) | 1 µL | 1.0 U / 50 µL |
| Total reaction volume | 50 µL | - |
* Do not use dNTPs from other kits or companies.
PCR CYCLE CONDITIONS
APPLICATION DATA
Example 1.Amplification of a GC-rich target
The GC rich target, lipase gene (Pseudomonas sp.) [GC content = 70%], was amplified using various PCR enzymes. KOD -Plus- could amplify the target gene without additives such as DMSO or PCR enhancer.
- Target
- Lipase gene (Pseudomonas sp.) [GC=70%]
- Reaction condition
- see typical reaction setup
- Sample
- Plasmid clone 10 ng / 50 µL reaction
- Cycling condition
1: KOD -Plus-
2: Taq DNA polymerase
3: Taq DNA polymerase + 5% DMSO
4: Taq DNA polymerase + PCR ehnancer (company A)
5: High efficient Taq DNA polymerase (company B)
M: 200 bp Ladder Markers
Example 2.Amplification of long targets
Long targets ranging in size from 0.6 to 3.6 kb were amplified using high fidelity PCR enzymes. All targets could be amplified by KOD -Plus-.
- Target
- Human β-globin gene
- Reaction condition
- see typical reaction setup
- Sample
- Plasmid clone 50 ng / 50 µL reaction
- Cycling condition
1: human β-globin 0.6 kb
2: human β-globin 1.3 kb
3: human β-globin 2.8 kb
4: human β-globin 3.6 kb
REFERENCES
- M. Takagi, M. Nishioka, H. Kakihara, M. Kitabayashi, H. Inoue, B. Kawakami, M. Oka, and T. Imanaka, Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol., 63: 4504-10 (1997)
- H. Hashimoto, M. Nishioka, S. Fujiwara, M. Takagi, T. Imanaka, T. Inoue and Y. Kai, Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. J Mol Biol., 306: 469-77 (2001)
- H. Mizuguchi, M. Nakatsuji, S. Fujiwara, M. Takagi and T. Imanaka, Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase. J Biochem., 126: 762-8 (1999)
- S. Fujii, M. Akiyama, K. Aoki, Y. Sugaya, K. Higuchi, M. Hiraoka, Y. Miki, N. Saitoh, K. Yoshiyama,K. Ihara , M. Seki, E. Ohtsubo and H. Maki, DNA replication errors produced by the replicative apparatus of Escherichia coli. J. Mol. Biol., 289: 835-850 (1999)