Description

KOD FLEX™ PCR Master Mix is a highly efficient and high-success-rate 2× PCR master mix. It incorporates a modified KOD DNA polymerase (UKOD), an elongation accelerator, and an unique buffer technology to enable high-efficiency, low-bias amplification of hard-to-amplify targets.

Efficient amplification is possible even for hard-to-amplify targets such as high GC/AT content regions, long DNA fragments, and those with low amplification specificity. Amplification is possible even from crude samples such as biological samples.

This master mix demonstrates approximately 11 times the fidelity of Taq DNA polymerase, making the resulting amplified products suitable for conventional sequencing analysis via cloning. Furthermore, it is characterized by reduced amplification bias related to sequence composition or amplicon size, making it suitable for library amplification in next-generation sequencing.

KOD FLEX™ PCR Master Mix contains two types of anti-KOD DNA polymerase antibodies that inhibit the polymerase and its 3ʹ→5ʹ exonuclease activity, thus allowing for Hot Start PCR. Furthermore, KOD FLEX™ PCR Master Mix generates blunt-end PCR products because of its 3ʹ→5ʹ exonuclease (proof-reading) activity.

Features

• High success-rate・High elongation efficiency
Original buffer composition enables high specificity. PCR efficiency is improved by adding an “Elongation Accelerator”. The extension rate can be reduced by decrements of up to 15 s/kb in singleplex PCR. (Note that to realize sufficient amplification, the extension rate of ~30–60 s/kb is recommended for amplifications of crude samples or for the multiplexed amplification of long targets.)

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

• Applicable for amplification from crude samples
KOD FLEX™ PCR Master Mix is effective for amplification from crude samples (e.g. biological samples, foodstuffs, soil extract, etc) because of its tolerance of the PCR inhibitors commonly present in crude samples.
• Homogeneous amplification (Low bias)
Homogeneous multiplex PCR of targets up to 10 kb can be achieved. Various regions of the genome or transcriptome can be amplified homogeneously even if these regions contain GC bias. The resulting amplicons are also suitable for next-generation sequencing analyses.
Effect of GC bias on amplification is minimized, enabling uniform amplification regardless of the target. This master mix is suitable for library amplification for next-generation sequencers of long reads as well as short reads due to low bias and high elongation.
• High fidelity
KOD FLEX™ PCR Master Mix exhibits approximately 11-fold higher fidelity than Taq DNA polymerase, so this master mix can accurately amplify long targets. The amplified products can be used for various purposes, including next-generation sequencing analysis as well as cloning.
• Primers containing uracil (U) or Inosine (I) can be used
KOD FLEX™ PCR Master Mix can use primers containing inosines (I) or uracil (U), whereas conventional high-fidelity PCR enzymes cannot. Bisulfite-treated targets and degenerate primers can be used.

Application

・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.

・High fidelity PCR

Specification

Category	Details
Source	E. coli strain carrying the cloned UKOD DNA polymerase gene
Storage condition	Store at -20°C

Components

KOD FLEX™ PCR Master Mix includes the following components for 200 reactions, 50 μl total reaction volume.

Category	Details
2x KOD FLEX™ PCR Master Mix	1 mL x 5

Application Data

Example 1.Amplification of long targets

Long targets were amplified using human genomic DNA as template.
Even extremely long targets of up to 40 kb were successfully amplified with KOD FLEX™ PCR Master Mix.

Example 2.Amplification of GC/AT-rich targets

Using human genomic DNA as template, GC-rich (approximately 70% G+C) and AT-rich (approximately 30% A+T) regions were amplified.
KOD FLEX™ PCR Master Mix demonstrated stable amplification of regions with high GC content as well as of regions with high AT content.

Example 3.Amplification from crude samples (blood)

The WT-1 gene (2.5 kb) was amplified using blood as template.
Using KOD FLEX™ PCR Master Mix, amplification was achieved even when 30% blood was added to the reaction mixture, without any inhibitory effects from the blood.

Example 4.Amplification of various targets

Eight different targets were amplified using human genomic DNA as template.
While no amplification or non-specific amplification was observed using other companies' reagents, good amplification was observed when using KOD FLEX™ PCR Master Mix.

Example 5.Multiplex PCR

Multiplex PCR was performed using human genomic DNA as template. The number of multiplex targets was gradually increased, starting from short fragments, to evaluate both reaction efficiency and specificity.
While some targets failed to amplify with other companies' reagents, KOD FLEX™ PCR Master Mix successfully produced bands corresponding to the intended number of multiplex targets.

Example 6.NGS library amplification

Genomic NGS libraries of E.coli (approximate GC content: 51%) and Thermus thermophilus (Tth, approximate GC content: 70%) were prepared, and those amplified with KOD FLEX™ PCR Master Mix for 7 cycles were compared with PCR-free libraries. Sequencing was performed using an Illumina MiSeq™ System.
Coverage comparisons across a range of GC contents showed no significant differences between PCR-free and amplified libraries, indicating that the libraries were successfully amplified without any bias.

Documents

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KOD FLEX PCR Master Mix-Toyobo