Home > Technology > High Fidelity & Efficient PCR Enzyme: KOD DNA polymerase

High Fidelity & Efficient PCR Enzyme: KOD DNA polymerase


The hyperthermophilic archaea Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) (Fig. 1) was isolated from a solfataric hot spring (Fig. 2) at Kodakara island (Fig. 3) in Japan, and was identified and characterized.

A family B DNA polymerase (KOD DNA polymerase) was found in this KOD1 strain and shows various unique properties (1).


Fig. 1. Thermococcus kodakaraensis KOD1


KOD DNA polymerase exhibits strong 3'→5' exonuclease activity (proof-reading activity), an activity that Taq DNA polymerase lacks.

Moreover, this enzyme exhibits excellent processivity and elongation capability, showing a five-fold higher extension rate (100-130 nucleotides/second) and 10-15-fold higher processivity (>300 bases) than that from Pyrococcus furiosus (Pfu DNA polymerase). The elongation rate of this enzyme is approximately 2 times higher than that of Taq DNA polymerase (Table 1)(Fig. 4) (1).


Fig. 2. Solfatara (Kodakara island, Japan)



Fig. 3.
Kodakara island (Kagoshima prefecture, Japan)


Table 1. Properties of thermostable DNA polymerases

Property Value for indicated DNA polymerase
KOD DNA polymerase Pfu DNA polymerase Taq DNA polymerase
Origin Archaea Archaea Bacteria
Deduced molecular mass (kDa) 90.0 90.1 93.9
Optimum temperature (ºC) 75 75 75
Optimum pH at 75ºC 6.5 6.5 8.0-8.5
Thermostability (half-life) 95ºC,12 h; 100ºC,3.0 h 95ºC, 6h; 100ºC, 2.9h 95ºC, 1.6 h
5'→3' exonuclease activity - - +
3'→5' exonuclease activity + + -
Terminal transferase activity - - +
Processivity (bases) >300 <20 ND
Elongation rate (bases/s) 106-138 25 61


Fig. 4. Comparison of elongation rates of KOD Pfu and Taq DNA polymerase.

The elongation rate was measured according to the length of synthesized DNA, using M13 ssDNA as the template at 75ºC.


In parallel with the study of the activities of KOD DNA polymerase, neutralization antibodies for the polymerase and proofreading activities were developed (2). These antibodies can be applied to the "hot start PCR technology" using KOD DNA polymerase.

The 3-D structure of DNA polymerase was characterized in 2003 (Fig. 5) (3).


Fig. 5. 3-D structure of KOD DNA polymerase

J. Mol. Biol., 306: 469-477 (2001)


KOD -Plus- (Code No. KOD-201) was developed based on KOD DNA polymerase and exhibits high PCR fidelity (Table 2).


Table 2. Comparison of the mutation frequency of each PCR enzyme.


Total bases Sequenced Number of mutated bases Mutation frequency (x10-5)
KOD -Plus- 145,753 5 3.4
KOD FX 144,535 19 13.1
Pfu DNA polymerase 113,080 12 10.6
Taq-base long-PCR enzyme 167,343 218 130.3
Taq DNA polymerase 102,708 145 141.2

Fidelity was measured as the mutation frequency by sequencing the PCR product. After cloning the PCR product (2.4kb of the human beta-globin region), about 96 clones were selected and sequenced.


KOD FX (Code No. KFX-101) was developed based on KOD DNA polymerase and shows a much greater PCR success-rate (based on efficiency and elongation capabilities) than KOD -Plus- (Code No. KOD-201) or other Taq-based PCR enzymes. KOD FX is also effective for amplification from crude specimens (e.g. mouse tail lysate, cultured cells).



Figure 6. Direct amplification from a crude mouse tail lysate

1,2: KOD FX
3~6: Other company's enzymes
M: Markers

References

1)

Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T.,
Appl Environ Microbiol., 63: 4504-10 (1997)


2)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126: 762-8 (1999)


3)

Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J Mol Biol., 306: 469-77 (2001)