DESCRIPTION

KOD FX Neo is based on the DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1(1) (2). KOD FX Neo provides greater efficiency and elongation capabilities than conventional PCR enzymes. In particular, KOD FX Neo shows greater amplification success from crude specimens.
KOD FX Neo is the improved version of the previous KOD FX (Code No. KFX-101). This product contains a unique "elongation enhancer" that suppresses the "plateau effect", enabling greater elongation rates and capabilities.
The KOD FX Neo enzyme solution contains two types of anti-KOD DNA polymerase antibodies that inhibit the polymerase and 3'→5' exonuclease activities, thus allowing for Hot Start PCR(3). KOD FX Neo generates blunt-end PCR products because of its 3'→5' exonuclease (proof-reading) activity.

FEATURES

  • KOD -Plus- Neo exhibits 80-fold greater PCR fidelity than Taq DNA polymerase.
  • "Elongation enhancer" enables greater amplification efficiency and elongation capability (up to 24 kb from human genomic DNA) compared to conventional PCR.
  • Requires only 30 sec/kb for the PCR extension step.
  • 2-step cycle conditions can be used for amplification using ≥ 20 mer primers (melting temperatures, Tm >63ºC).

APPLICATIONS

  • Genotyping (Amplification from crude sample)
    Direct amplification from;
    -Whole blood, cultured cell, mouse tail, mouse toe , plant and food stuff etc.
    -Various kinds of lysate from mouse tail, plant, fish fin, soil etc.
    - Gram-positive bacteria, fungus, yeast etc.
  • Efficient amplification of difficult targets (high G/C, long ) with fast mode.
  • FHigh 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, 50 µl total reaction volume:.

KOD FX Neo (1.0 U / µl) * 200 µl × 1
2 x PCR Buffer for KOD FX Neo** 1.7 ml × 3
2 mM dNTPs 1 ml × 2

*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize the polymerase and 3'→5' exonuclease activities.
** 2x PCR Buffer for KOD FX Neo is a liquid (not frozen) when stored at -20ºC. Although it does freeze below -20ºC, the quality is not affected.

TYPICAL PCR REACTION SETUP

Component Volumes Final Concentration
PCR grade water X µl 1x
2x PCR buffer for KOD FX Neo 25 µl 1x
2mM dNTPs* 10 µl 0.4 mM each
10pmol /µl Primer #1 1.5 µl 0.15–0.3 µM
10pmol /µl Primer #2 1.5 µl 0.15–0.3 µM
Template DNA Y µl

Genomic DNA ≤ 200 ng/50 µl

Plasmid DNA ≤ 50 ng/50 µl

cDNA ≤ 200 ng(RNA equiv.)/50 µl

Crude sample ≤0.5–2 µl/50 µl (see [ 7 ])

KOD FX Neo (1.0U/µl) 1 µl 1.0 U / 50 µl
Total reaction volume 50 µl  

*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize the polymerase and 3'→5' exonuclease activities.
** 2x PCR Buffer for KOD FX Neo is a liquid (not frozen) when stored at -20ºC. Although it does freeze below -20ºC, the quality is not affected.

PCR CYCLE CONDITIONS

APPLICATION DATA

Example 1.Amplification of long targets

Targets of various sizes were amplified from human genomic DNA by several PCR enzymes according to the recommended conditions of each enzyme. KOD FX Neo successfully amplified targets up to 40 kb.

Template
Human genomic DNA 200ng/50µl

M : λ/Hin d III Marker
1:tPA 24kb
2:HBg 32kb
3:HBg 40kb

Example 2.Amplification from mouse tail lysates

The amplification efficiency was then compared between various PCR enzymes using mouse tail lysates as templates. KOD FX Neo showed greater amplification efficiency than the other enzymes.

Template
mouse tail lysate (Alkaline lysis mehod) 0.25µl

1:Mouse TATA box binding protein (TBP) 0.5 kb
2:Mouse transferrin receptor (Tfr) 1.5 kb
3:Mouse membrane glycoprotein (Thy-1) 2.6 kb
M:100 bp DNA Ladder

Example 3.Amplification from leaf specimens

Two targets (2.2 and 4.6 kb) were amplified using lysates from tobacco leaves. Each PCR reaction was performed according to the recommended conditions with 35 cycles. KOD FX Neo showed greater amplification from lysates prepared by the "one-step method".
Various targets were then directly amplified using small pieces of tobacco leaves (2 × 2 mm). KOD FX Neo successfully amplified DNA using these templates.

Template
Lysate* [Tobacco] 1µl
* One-step method
Target
ribulose-1,5-bisphosphate carboxylase / oxygenease large subunit
N-methyltransferase gene (rbcmtT)

Template
Tobacco leaf (2 × 2 mm)
Target
1:ribulose-1,5-bisphosphate carboxylase / oxygenease large subunit gene (rbcL) 1.3 kb
2:rbcmtT 2.2 kb
3:rbcmtT 4.6 kb
M: 1 kb DNA Ladder

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)