DESCRIPTION

KOD OneTM PCR master Mix and KOD OneTM PCR Master Mix -Blue- are 2 x PCR master mixes based on genetically modified KOD DNA polymerase (UKOD). KOD OneTM series enables fast PCR, which has an extension time of 5 sec/ kb by applying UKOD and a new Elongation Accelerator. In addition, these master mixes provide greater efficiency and elongation capabilities than conventional PCR enzymes. In particular, these show greater amplification success from crude specimens. Furthermore, these master mixes can be applied to amplify from templates containing uracils (dU) or using primers containing inosines (dI) and uracils (dU).

KOD OneTM series contains two types of anti-KOD DNA polymerase antibodies that inhibit the polymerase and 3’→5’ exonuclease activities, thus allowing for Hot Start PCR. These master mixes generate blunt-end PCR products because of 3’ → 5’ exonuclease (proof-reading) activity of KOD DNA polymerase.

FEATURES

- Fast
KOD OneTM series can amplify the targets using the following very short conditions:

  • ≤1 kb:　1 sec
  • 1~ 10 kb:　5 sec/ kb
  • 10 kb~:　10 sec/ kb

The cycling conditions can be set flexibly when various targets having different sizes are amplified.

- Easy to Use
KOD OneTM series contains all reaction components except primers and templates and provide high reproducibility by reducing operations. In addition, KOD OneTM PCR Master Mix -Blue- includes a loading dye (BPB) to allow direct loading onto agarose gels.

- High Fidelity
KOD OneTM series exhibits approximately 80-fold higher fidelity than Taq DNA polymerase. These mixes can be used for various purposes where this would be an advantage (e.g., in the preparation of long target amplicons for sequencing).

- High Efficiency
KOD OneTM series is effective for amplification from crude samples (e.g., biological samples, foodstuffs, soil extract, etc.). Various samples or lysates can be used directly as templates.

- Primers or Templates Containing Inosines (dI) or Uracils (dU) Can Be Used
KOD OneTM series can use primers or templates containing inosines (dI) or uracils (dU), whereas conventional high-fidelity PCR enzymes cannot.

APPLICATIONS

  • Direct PCR
  • Colony PCR
  • Amplification of NGS libraries
  • Site direct gene mutation

SOURCE

E. coli strain carrying the cloned UKOD DNA polymerase gene

STORAGE CONDITION

Store at -20°C ( 4°C for a month )

COMPONENTS

KOD OneTM series include the following components for 200 reactions, 50 μl total reaction volume.

<KMM-101>

KOD OneTM PCR master Mix 1 mL x 5

<KMM-201>

KOD OneTM PCR master Mix -Blue- 1 mL x 5

Note
The reagents can be stored at 4°C for a month. For longer storage, the reagents should be kept at -20°C.

APPLICATION DATA

Example 1.Fast PCR

Various targets were amplified with KOD OneTM PCR Master Mix and KOD OneTM PCR Master Mix -Blue- using the ultra fast cycling conditions. KOD OneTM series successfully amplified all targets.

Reaction Mix
PCR cycle

Example 2.Amplification from cDNA

Inhibitory effect of RNA in cDNA was compared using various PCR enzymes. KOD OneTM Master Mix was not susceptible to RNA inhibition, and it was able to amplify targets under high concentrations of cDNA.

Reaction Mix
PCR cycle

Example 3.PCR error ratio

The error ratio of various PCR enzymes were compared by determining the sequences of the amplicons from human β-globin gene. The amplicons were cloned into the vector using TArget cloneTM -Plus- (Code No. TAK-201) and the sequences were determined.
KOD OneTM PCR Master Mix and KOD OneTM PCR Master Mix -Blue- showed excellent fidelity and the mutation frequency were approximately 80 times lower than Taq DNA polymerase.

Example 4.Amplification using degenerate primers, containing inosine(I)

The 2.8 kb fragments were amplified using degenerate primers containing inosine(I). KOD OneTM PCR Master Mix was able to amplified, whereas conventional high-fidelity PCR enzymes cannot.

Reaction Mix
PCR cycle
Primer sequence
Fwd: ATGGTICARATHCCICARAAY
Rev: RTGIGCYTGRTCCCARTTYTC

Example 5.Amplification from crude samples

Amplification from whole blood and that from mouse lysate were compared. KOD OneTM PCR Master Mix amplified the targets efficiently.

Reaction Mix
PCR cycle

Example 6.Amplification of NGS library -Minimal amplification bias-

A NGS library of Thermus thermophilus genome (GC content 70%) was amplified using KOD OneTM PCR Master Mix and another company's product, and the amplified libraries were sequenced on an Illumina MiSeq. This is the result of examining the GC bias of amplified libraries. The Normalized Coverages (Blue plot) close to 1 indicate low bias. KOD OneTM PCR Master Mix showed the lower GC bias than another company's product.

GC bias plots were generated, with %GC content of 100 bp windows on the X axis. Normalized coverage is indicated by the blue circles (〇), %GC of the reference sequence indicated by the red lines (-) and base quality at %GC indicated by the green line (-).

Reaction Mix
PCR cycle
Primer sequence
Fwd: AATGATACGGCGACCACCGAGATC
Rev: CAAGCAGAAGACGGCATACGAG

Please see below for library input amounts and cycle numbers.
(The optimal number of cycles may vary from 1 to 3 cycles depending on the sample type and mold size distribution.)

library input amount number of cycle
1 μg 0 - 1
500 ng 1 - 2
100 ng 4 - 5
50 ng 5 - 6
10 ng 8 - 10
1 ng 13 - 15
0.25 ng 16 - 18