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High Success-rate DNA polymerase
KOD FX
Code No. KFX-101
DESCRIPTION
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KOD FX is based on DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1(1)(2). KOD FX results in much greater PCR success based on efficiency and elongation capabilities than KOD -Plus- or other Taq-based PCR enzymes. KOD FX enzyme solution contains two types of anti-KOD DNA polymerase antibodies that inhibit polymerase and 3'→5' exonuclease activities, thus allowing for Hot Start PCR(3). KOD FX generates blunt-end PCR products, due to 3'→5' exonuclease (proof-reading) activity.
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Features
- ・Effective for the amplification of GC-rich targets and crude samples.
- ・Hotstart technology enables highly efficient amplification.
- ・KOD FX enables the following amplifications (Maximum):
- ・40kb from lambda phage DNA, 24kb from human genomic DNA, and 13.5kb from cDNA.
- ・The PCR error ratio is about 10 times less than that of Taq DNA polymerase.
Amplification from a crude mouse tail lysade
1,2:KOD FX
3~6: Other comparty's enzymes
M: Markers
Table I. Comparison of the mutation frequency of each PCR enzyme.
| Total bases Sequenced | Number of mutated bases | Mutation frequency (x10-5) | |
|---|---|---|---|
| KOD FX | 144,535 | 19 | 13.1 |
| KOD -Plus- | 145,753 | 5 | 3.4 |
| 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 a mutation frequency by sequencing the PCR product. After cloning the PCR product
(2.4kb of the human bata-globin region), about 96 clones were selected and sequenced.
Details
APPLICATIONS
- High success-rate 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;
| KOD FX(1.0 U/µL)* | 200 µL × 1 |
| 2 × PCR Buffer for KOD FX** | 1.7 µL × 3 |
| 2mM dNTPs | 1 µL × 2 |
*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3'→5' exonuclease activities.
** 2× PCR Buffer for KOD FX is a liquid (not congealed) when in storage at -20°C. Although it does congeal below -20°C, the quality is not affected.
TYPICAL PCR REACTION SETUP
| Component | Volumes | Final Concentration |
|---|---|---|
| 2x PCR buffer for KOD FX | 25 µL | 1× |
| 2 µM dNTPs* | 10 µL | 0.4 µM each |
| 10 pmol / µL Primer #1 | 1.5 µL | 0.3 µM |
| 10 pmol / µL Primer #2 | 1.5 µL | 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 FX (1.0U/ µL) | 1 µL | 1.0 U / 50 µL |
| Total reaction volume | 50 µL |
PCR CYCLE CONDITIONS
-Extension time should be set at 1 min. per 1kbp of target length. Although even 30 sec./ kb will give amplification in many
cases, amplification efficiency or reliability may be decreased (See Example 3).
-Because this enzyme possesses an extremely high amplification efficiency, 25~30 cycles usually give sufficient amplification. For a low-copy number target, or over 10 kb target length, 30~40 cycles are recommended.
-The step-down cycle condition is effective for trouble shooting a smear amplification in a long-distance PCR(>10kb).
Application Data
Example 1.Amplification from a crude mouse tail lysate
- Target
- Mouse membrane glycoprotein (Thy-1) gene 2.6kb <M10246>
- Reaction condition
- see typical PCR reaction setup
- Sample
- Mouse tail lysate 0.5µL / 50µL reaction
- Cycling condition
1,2: KOD FX
3,4: Taq based PCR enzyme (Company A)
5,6: Taq based PCR enzyme (Company B)
M: Markers
[Preparation of mouse tail lysates (Alkaline lysis method)]
Mouse tail (ca. 3 mm)
↓←50 mM NaOH 180 µL
Vortex well
↓95°C, 10 min.
↓←1M Tris-HCl (pH8.0) 20 µL
Vortex well
↓12,000 rpm, 10 min.
Supernatant
Mouse membrane glycoprotein (Thy-1) gene was amplified using various PCR enzymes from a mouse tail lysate prepared by the alkaline lysis method. As a result, KOD FX could successfully amplify the target whereas the other PCR enzymes gave no amplification bands.
Example 2.Amplification of a GC-rich target
The GC-rich target, human IGF2R (8.9 kb) was amplified using various PCR enzymes. This gene contains very high GC-rich regions (CG content = 90%). KOD FX successfully amplified the target.
- Target
- Human IGF2R (8.9kb) contains 90% GC region
- Reaction condition
- see typical PCR reaction setup
- Sample
- cDNA from 50 ng total RNA of HeLa cells
- Cycling condition
1: KOD FX
2~8: Other company's enzymes
M1: 1 kb Ladder Markers
M2: λ/HindIII Markers
Example 3.Amplification from crude samples
Various lengths of β-globin targets were amplified from cultured cells (Jurkat cells). The target genes (1.3, 3.6, and 8.5 kb) were successfully amplified using KOD FX.
- Target
- Human β-globin gene (1.3 kb, 3.6 kb, 8.5 kb)
- Reaction condition
- see typical PCR reaction setup
- Sample
- Jurkat cells (2x104 cells/ 50mL reaction mixture)
- Primer condition:
- 1.3kb F primer:5'-TTAGGCCTTAGCGGGCTTAGAC-3'
1.3kb R primer:5'-CCAGGATTTTTGATGGGACACG-3'
3.6kb F primer:5'-GGTGTTCCCTTGATGTAGCACA-3'
3.6kb F primer:5'-ACATGTATTTGCATGGAAAACAACTC-3'
8.5kb F primer:5'-TGATAGGCACTGACTCTCTGTCCCTTGGGCTGTTT-3'
8.5kb F primer:5'-ACATGATTAGCAAAAGGGCCTAGCTTGGACTCAGA-3 - Cycling condition
Example 4.Amplification of long targets
The long-target amplification capability of KOD FX was evaluated by amplifying β-globin genes ranging in size from 1.3 to 17.5 kb. As a result, distinct bands were successfully amplified from genomic DNAs by KOD FX.
- Target
- Human β-globin gene (1.3 kb, 3.6 kb, 8.5 kb, 17.5 kb, 24 kb)
- Reaction condition
- see typical PCR reaction setup
- Sample
- 50~200 ng / 50 mｌ reaction mixture
- Cycling condition
M1：1kb DNA ladder
M2：λ/ Hind III digest
REFERENCES
- Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)
- Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J Mol Biol., 306: 469-77 (2001)
- Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126: 762-8 (1999)