DESCRIPTION

KOD Dash is a highly efficient DNA polymerase mixture developed based on the Barns' method(1). This method uses a DNA polymerase which lacked a 3'→5' exonuclease (proofreading) activity and a small amount of an archaeal DNA polymerase with proofreading activity. In the reagent, the 3'→5' exonuclease activity-deficient mutant <KOD EXO(-)> of KOD DNA polymerase(2) and KOD DNA polymerase are used. Because the proofreading activity repairs misincorporated nucleotide bases causing the termination of a polymerase reaction, PCR with this mixed enzyme solution enables highly efficient amplification.
KOD Dash generates dA overhang-ended PCR products. Therefore, the PCR products can be cloned using a standard TA cloning method.

FEATURES

  • Effective for the amplification of various targets from a small starting template amount.
  • Shows greater elongation velocity than Taq DNA polymerase (2 folds) and Pfu DNA polymerase (6 folds) due to the intrinsic property of KOD DNA polymerase.
  • The PCR error ratio of this enzyme mixture is approximately 3 to 4 times less than that of Taq DNA polymerase.

APPLICATIONS

  • Fast PCR

SOURCE

E. coli strain carrying the cloned KOD DNA polymerase gene and KOD exo (-) 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, 50 mM KCl, 1 mM DTT, 0.1 % Tween 20, 0.1% Nonidet P-40, 50% Glycerol Store at -20ºC

COMPONENTS

The provided reagents include the following components for 200 reactions:

KOD Dash (2.5 U/µl)100 µl
10x PCR Buffer for KOD Dash* 1.2 ml
2 mM dNTPs 1.0 ml

PRINCIPLE

TYPICAL PCR REACTION SETUP

Component Volumes Final Concentration
10x Buffer for KOD Dash 5 µl
2 mM dNTPs* 5 µl 0.2 µM each
10 pmol/µl Primer #1 1 µl 0.2 µM
10 pmol/µl Primer #2 1 µl 0.2 µM
Template DNA X µl Genomic DNA 10-1000 ng/50 µl
Plasmid DNA 1-50 ng/50 µl
cDNA ~1 mg (RNA equiv.)/50 µl
PCR grade water Y µl  
KOD Dash (2.5 U/µl) 1 µl 1.25 U / 50 µl
Total reaction volume 50 µl  

* Do not use dNTPs from other kits or companies.

PCR CYCLE CONDITIONS

*For colony-direct PCR, the pre-denaturation step should be set for 4 min.
**Extension time should be set at 30 s per 1 kb of target length.

APPLICATION DATA

Example 1.Detection of HCV using the fast cycle

The sensitivities of PCR of KOD Dash and Taq DNA polymerase were compared using a normal cycle and fast cycle. HCV RNA from serum specimens was detected using the nested RT-PCR method reported by Okamoto et al. [Japan. J. Exp. Med. 60: 215-222 (1990)]. KOD Dash can rapidly detect low copies of HCV in serum specimens.

Template
HCV cDNA synthesized from HCV RNA isolated from serum specimens
Target
HCV pre-core gene
PCR cycling condition

PCR cycling condition

M: φX174/Hinc II digest
1: 2.5 X 103 copies/reaction
2: 2.5 X 102 copies/reaction
3: 2.5 X 10 copies/reaction
4: 2.5 copies/reaction

Example 2.Colony-direct PCR using E. coli cells

DNAs inserted in vectors were directly detected using E. coli cells carrying the cloned transferrin receptor gene (4.5 kb). The cloned transferrin receptor gene was efficiently detected using a fast PCR cycle.

Template
Colony of E. coli (JM109) carrying the cloned transferrin receptor gene in the vector
Target
Transferrin receptor gene 4.5 kb
PCR cycling condition

REFERENCES

  • W.M. Barns, PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage
    templates.Proc. Natl. Acad. Sci. USA, 91: 2216-2220 (1994)
  • 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)