KOD -Plus- Neo is based on a DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1(1) (2). This polymerase exhibits excellent PCR fidelity because of its efficient 3'→5' exonuclease activity (proof reading activity). This product contains a unique "elongation enhancer" that suppresses the "plateau effect" produced by conventional PCR. Therefore, this reagent exhibits greater amplification efficiency and elongation capability compared to the previous version of KOD -Plus- (Code No. KOD-201).
Moreover, this enzyme requires only 30 sec/kb for the PCR extension step. This facilitates the long range PCR.
This enzyme contains two anti-KOD DNA polymerase antibodies that inhibit polymerase and 3'→5' exonuclease activity, thus allowing for Hot Start PCR(3). This polymerase generates blunt-end PCR products due to 3'→5' exonuclease (proof-reading) activity.
- 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).
（Number of misincorporated bases/100,000 bases）
*Mutation frequency was measured by sequence analysis of human β-globin gene products amplified from human genomic DNA via TA cloning.
- High fidelity PCR
- Fast and efficient PCR
E. coli strain carrying the cloned KOD DNA polymerase gene
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.
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
This reagent includes the following components for 200 reactions, 50 µl total reaction volume:.
|KOD -Plus- Neo (1.0 U / µl)*||200 µl × 1|
|10 x PCR Buffer for KOD -Plus- Neo||1 ml × 1|
|25 mM MgSO4||1 ml × 1|
|2 mM dNTPs||1 ml × 1|
*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3'→5' exonuclease activity.
TYPICAL PCR REACTION SETUP
|10x Buffer for KOD -Plus- Neo||5 µl||1x|
|2mM dNTPs*||5 µl||0.2 mM each|
|25mM MgSO4||3 µl||1.5 mM|
|10 pmol/µl Primer #1||0.75–1.5 µl||0.15–0.3 µM|
|10 pmol/µl Primer #2||0.75–1.5 µl||0.15–0.3 µM|
|Template DNA||X µl||Genomic DNA ≤ 200 ng/50 µl
Plasmid DNA ≤ 50 ng/50 µl
cDNA ≤ 200 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
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 -Plus- Neo successfully amplified targets up to 17.5 kb.
【Cycling conditions of KOD - Plus- Neo】
1: Human β-globin (1.3 kb)
2: Human β-globin (2.7 kb)
3: Human β-globin (3.6 kb)
4: Human β-globin (8.5 kb)
5: Human β-globin (17.5 kb)
M: 1 kb DNA Ladder
Example 2.Amplification of various protein kinase targets
Various protein kinase open reading frames (ORFs) were amplified using cDNA synthesized from total RNA of HeLa cells. KOD -Plus- Neo successfully amplified all targets.
【Cycling condition of KOD - Plus- Neo】
1: Human brk Tyrosine kinase (1.6 kb)
2: Human rac protein kinase-alpha (1.7 kb)
3: Human 63kDa protein kinase (2.0 kb)
4: Human c-syn protooncogene (2.2 kb)
5: Human FER Tyrosine kinase (2.7 kb)
6: Human cell adhesion kinase beta (3.2 kb)
7: Human Jak2 kinase (3.6 kb)
M: 1 kb DNA Ladder
- 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)