KOD -Plus- Mutagenesis Kit
Site-directed mutagenesis kit
This kit is an inverse PCR (iPCR)-based site-directed mutagenesis kit using KOD DNA polymerase (1)( 2) as a high-fidelity PCR enzyme. This reagent was developed based on a high efficient and efficient PCR reagent, "KOD-Plus- (Code No. KOD-201)", which consists of KOD DNA polymerase and anti-KOD DNA polymerase antibodies(3) for Hot Start PCR.
This kit enables not only the introduction of point mutations, but also the introduction of large insertions and deletions. The PCR fidelity of KOD-Plus- is greater than Taq DNA polymerase (ca. 80-fold); therefore, unexpected, 2nd-site mutations can be reduced. PCR reactions can be performed using standard PCR primers and do not require phosphorylated primers, because this protocol contains a 'Phosphorylation Step' of PCR products.
Features
- Applicable for various mutations, such as substitutions, insertions, and deletion mutations.
- Substitution (e.g. Point mutation, Point mutation library)
- Deletion (e.g. 1 bp – several kbp deletion)
- Insertion (e.g. Introduction of His-tag sequence)
- High efficiency (95% maximum) can be obtained.
- Simple protocol facilitates speedy experiments. Phosphorylated primers are not required
Principle
Mutation Introduction
- Inverse PCR of plasmid DNA, using a mutation primer.
- Plasmid DNA is digested by Dpn I.
Note: Dpn I digests methylated DNA, such as plasmid DNA from typical E. coli cell lines (e.g. JM109 and DH5α). - Self-ligation of PCR products is performed by a reaction with T4 polynucleotide kinase and ligase.
- Transformation of E. coli cell lines using self-ligated PCR products.
Components
| KOD-Plus-(1 U/µL) | 25 µL |
|---|---|
| 10x Buffer for iPCR | 125 µL |
| 2 mM dNTPs | 125 µL |
| DpnI (10 U/µL) | 50 µL |
| T4 Polynucleotide Kinase (5 U/µL) | 50 µL |
| Ligation high (T4 Ligase + Buffer Mixture) | 250 µL |
| Control Plasmid pAK119M (50 ng/µL) | 10 µL |
| Control Primer #1 (10 pmol/µL) | 10 µL |
| Control Primer #2 (10 pmol/µL) | 10 µL |
High Efficiency
Efficiency Comparison of Deletion and Insertion Mutations
Using the 7.3 kb plasmid FLJ32066, mutation efficiacny was eveluated :a 90‑base deletion and an 18‑baseinsertion correspinding to 6xHis were compared using this product and Company A’s kit. This product consistently generated the desired mutants with high efficiency, even for mutation types such as deletions and insertions where other companies’ kits often show reduced performance.
The result of 90 base deletion
| Number of sequenced clones |
Number of clones that had the desired mutation |
Success rate | |
|
KOD Plus |
16 | 14 | 88% |
| Company A's kit | 16 | 7 | 44% |
The result of 18 base insertion
| Number of sequenced clones |
Number of clones that had the desired mutation |
Success rate | |
|
KOD Plus |
16 | 15 | 94% |
| Company A's kit | 16 | 0 | 0% |
Evaluation of the insertion frequency of off-target (2nd-site) mutations
Mutagenesis was performed using the control plasmid (4.3 kb) provided with the kit as a template (with PCR conducted for 8 cycles). A 2,000 bp region was then sequenced for each of 24 clones obtained from the resulting mutants. The analysis revealed that, out of a total of 48,000 bases examined, only one unintended mutation was present. This result is equivalent to or better than that achieved with a non-PCR-based kit from Company A.
| Mutation Induction Rate |
Number of clones |
Total number of sequenced bases | Number of bases with 2nd-site mutations | Insertion rate of 2nd-site mutations (x10^-5) |
|
|
KOD Plus |
>80% | 24 | 48,000 | 1 | 2.08 |
| Company A's kit | >80% | 24 | 48,000 | 2 | 4.16 |
References
- Arai, S et al. Role of RIP140 in metabolic tissues: Connections to disease. FEBS Lett. 2007;581(29):5649–5657. doi: 10.1016/j.febslet.2007.11.017
- Torisu Y, et al. Human homolog of NOTUM, overexpressed in hepatocellular carcinoma, is regulated transcriptionally by β‑catenin/TCF. Cancer Sci. 2008;99(6):1139–1146. doi: 10.1111/j.1349-7006.2008.00814.x
- Shinmen N, et al.Functional conversion of fatty acyl-CoA synthetase to firefly luciferase by site-directed mutagenesis: A key substitution responsible for luminescence activity. FEBS Lett. 2009;583(12):1916–1922. doi: 10.1016/j.febslet.2009.05.018
Ordering Information
| Product | Storage | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|---|
| KOD -Plus- Mutagenesis Kit | -20℃ | SMK-101 | 20 Reactions | 357.00 | Buy |