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High Fidelity DNA polymerase

KOD -Plus-


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.

Table I. 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 definition

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 MgSO41.0 µl × 1
2 mM dNTPs1.0 µl × 1

*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3'→5' exonuclease activity.


Typical PCR reaction setup

Component
Volume Final Concentration
10x Buffer for KOD -Plus- 5 µl
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.

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

Target:Lipase gene (Pseudomonas sp.) [GC=70%]

Reaction condition: see typical reaction setup

Sample: Plasmid clone 10 ng / 50 μl reaction

Cycling condition:

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-.

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

Target: Human β-globin gene

Reaction condition: see typical reaction setup

Sample: Plasmid clone 50 ng / 50 μl reaction

Cycling condition:


References

1) 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)

2) 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)

3) 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)

4) 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)