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KODmutant高保真高效聚合酶高表达质粒载体菌种-BioVector NTCC保藏中心

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  • 货  号:KOD mutant DNA polymerase expression plasmid
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KOD mutant DNA polymerase聚合酶高表达质粒载体菌种


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

PropertyValue for indicated DNA polymerase
KOD DNA polymerasePfu DNA polymeraseTaq DNA polymerase
OriginArchaeaArchaeaBacteria
Deduced molecular mass (kDa)90.090.193.9
Optimum temperature (ºC)757575
Optimum pH at 75ºC6.56.58.0-8.5
Thermostability (half-life)95ºC,12 h; 100ºC,3.0 h95ºC, 6h; 100ºC, 2.9h95ºC, 1.6 h
5'→3' exonuclease activity--+
3'→5' exonuclease activity++-
Terminal transferase activity--+
Processivity (bases)>300<20ND
Elongation rate (bases/s)106-1382561


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 SequencedNumber of mutated basesMutation frequency (x10-5)
KOD -Plus-145,75353.4
KOD FX144,5351913.1
Pfu DNA polymerase113,0801210.6
Taq-base long-PCR enzyme167,343218130.3
Taq DNA polymerase102,708145141.2

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)



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