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

Application

High fidelity PCR

Specification

Table 1.Comparison of the mutation frequency of each PCR enzyme.

	Colony number		Mutation frequency (%)
	Total	Mutant	Mutation frequency (%)
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).

Category	Item	Details
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:

Item	Details
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)

Documents

Download Manual Download SDS Request COA

KOD -Plus- Mutagenesis Kit-Toyobo