DESCRIPTION

KOD -Multi & Epi- is a high-fidelity PCR enzyme based on genetically modified KOD DNA polymerase(UKOD). This modified enzyme enables amplification from templates containing uracils (U) or using primers containing inosines (I) and uracils (U). Furthermore, addition of the Elongation Accelerator significantly reduces amplification bias during PCR. KOD -Multi & Epi- can be applied to various purposes such as a) multiplex PCR, b) bisulfite PCR in epigenetics research, and c) metagenomics research. This enzyme is also applicable for the preparation of DNA fragments for next-generation sequencing and capillary sequencing via cloning because the enzyme exhibits about 11-fold higher PCR fidelity.
KOD -Multi & Epi- 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 -Multi & Epi- generates blunt-end PCR products because of its 3ʹ→5ʹ exonuclease (proof-reading) activity.

FEATURES

  • Homogeneous amplification (Low bias)
  • Effective amplification from templates containing uracils (U) and primers containing uracil (U) or Inosine (I) can be used
  • High fidelity
  • Applicable for amplification from crude samples
  • Highly efficiency

APPLICATIONS

SOURCE

E. coli strain carrying the cloned KOD DNA polymerase gene

STORAGE CONDITION

Store at -20℃

COMPONENTS

KOD -Multi & Epi-® (1.0 U/uL)* 200 uL × 1
2× PCR Buffer for KOD -Multi & Epi-** 1.7 ml × 3

*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize the polymerase and inhibit its 3ʹ→5ʹ exonuclease activities.
**The 2× PCR Buffer for KOD -Multi & Epi- contains dNTPs (dATP, dGTP, dCTP and dTTP), and Mg2+ at 4.0 mM (final concentration: 2.0 mM).

APPLICATION DATA

Example 1.PCR performance in Multiplex PCR

The small targets from 200 to 1,000 bp and the large target from 1 to 10 kb were amplified by single-plex or multiplex PCR. Each target was successfully amplified by both single-plex and multiplex PCR.

Sample
Human genomic DNA 50ng
Primer concentration
0.3mM each
Multiplex PCR cycle
94 C, 2 min
98 C, 10 sec
68 C, 30 sec (25 cycles)

M:100 bp DNA ladder
1:DDB2 200 bp
2:FANCG 250 bp
3:HBg 300 bp
4:CDH1 400 bp
5:chrome9 500 bp

6:ERCC4 550 bp
7:HRAS 600 bp
8:PRF1 700 bp
9:BRCA1 800 bp
10:CDK4 1000 bp
MP:Multiplex PCR 1~10

Sample
Human genomic DNA 50ng
Primer concentration
0.3mM each
Multiplex PCR cycle
94 C, 2 min
98 C, 10 sec
68 C, 5 min (25 cycles)

M:1 kb DNA ladder
1:chromo9 1 kb
2:MSH6 1.8 kb
3:BRCA2 2.3 kb
4:WT-1 2.5 kb
5:FNCE 3 kb

6:RAD51D 4 kb
7:KRAS 5 kb
8:BRCA1 7 kb
9:DDB2 10 kb
MP: Multiplex PCR 1~9

Example 2.Amplification from bisulfite-treated DNA

The target DNAs (917–1,583 bp) were amplified using bisulfite-treated CpG-methylated Jurkat genomic DNA. All targets were successfully amplified using KOD -Multi & Epi-®.

Bisulfite treatment
EpiTect Fast DNA Bisulfite Kit (Qiagen)
Primer concentration
0.3mM each
Sample
55 ng of bisulfite-treated DNA / 50mL reaction
Multiplex PCR cycle
94 C, 2 min
98 C, 10 sec
60 C, 30 sec
68 C, 30 sec/kb (40 cycles)

M: 100 bp DNA ladder
1:TGFb 917 bp
2:BRCA2 1134 bp
3:APOE 1487 bp
4:TGFb 1583 bp

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)