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High Success-rate DNA polymerase

KOD FX

Description

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.


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.

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.

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
 Volume 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

1,2: KOD FX

3,4: Taq based PCR enzyme (Company A)

5,6: Taq based PCR enzyme (Company B)

M: Markers

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:

[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.

1: KOD FX

2~8: Other company's enzymes

M1: 1 kb Ladder Markers

M2: λ/HindIII Markers

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:
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

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)Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J Mol Biol., 306: 469-77 (2001)


3)Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126: 762-8 (1999)