Site Directed Mutagenesis

Key Takeaways:

Site-directed mutagenesis (SDM) enables precise DNA sequence alterations.
Used for investigating protein activity changes and introducing beneficial mutations.
GenieClone kits facilitate efficient, multiple mutations in one reaction.
Challenges include primer design, template quality, and reaction optimization.

What is Site Directed Mutagenesis?

Site-directed mutagenesis (SDM), sometimes referred to as site-specific or directed mutagenesis, is a method of altering the nucleotide sequence of a gene at a specified location. SDM can be used to introduce specific nucleotide substitutions (or deletions) in a controlled manner. This method is in contrast to general mutagenesis which employs mutagenic compounds or high-energy radiation to randomly alter DNA. SDM is a highly versatile technology that can be used to probe gene function of an isolated gene product in a complex biological system. SDM can also be used to engineer proteins and even entire organisms for new functions or capabilities.

There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including:

To study changes in protein activity that occur as a result of the DNA manipulation.
To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property.
To introduce or remove restriction endonuclease sites or tags.

Assay Genie Site Directed Mutagenesis Kits

Product Code	Product Name
MORV0104 – 10 reactions MORV0105 – 25 reactions	GenieClone DNA Assembly Site-Directed Mutagenesis Kit v1
MORV0106 – 10 reactions MORV0107 – 25 reactions	GenieClone DNA Assembly Site-Directed Mutagenesis Kit v2

GenieClone DNA Assembly Site-Directed Mutagenesis Kit

GenieClone Site-Directed Mutagenesis Kit is a site-directed mutagenesis system based on GenieClone rapid cloning technology. This kit is designed for rapid site-directed mutagenesis of 3 to 5 separate mutations in the same reaction in less than 3 hours. Based on GenieClone rapid cloning technology, this kit uses homologous recombination to replace the conventional annealing ring-forming reactions meaning much less template and a more flexible primer design strategy.

With seamless splicing of the two PCR products by GenieClone technology, the kit can complete up to two separate mutations with a single amplification reaction. DpnI digested products of the specific amplicons can be directly added to the recombination reaction without purification. The highly optimized reaction buffer, fast protocol and superior site-directed mutagenesis efficiency make GenieClone Site-Directed Mutagenesis Kit v1 make an excellent choice for introducing 3 to 5 mutations at discontinuous sites simultaneously on the same plasmid.

Figure 1: Single base site-directed mutation using GenieClone Site-Directed Mutagenesis Kit v1

Site Directed Mutagenesis Kit - Product Advantages

Introduce 3 - 5 separate mutations in the same reaction.
Site-directed mutagenesis can be performed on up to 5 discontinuous sites (more than 50 bp apart) simultaneously on the target plasmid.
Single kit for cloning and mutagenesis.
Includes Genie Fusion Ultra High-Fidelity DNA Polymerase providing high-fidelity PCR with extremely low mutation rates & excellent long fragment amplification for any plasmid amplification up to 20 kb as well as GC-rich templates.
Amplification is carried out exponentially and the template usage is extremely low, which is beneficial to the complete degradation of the original methylation template.
DpnI eliminates contamination of the original template and decreases background.
Amplified products can be directly used in the recombination reaction after DpnI treatment.
The reaction mixture can be used to directly transform chemically competent E.coli cells.

Site Directed Mutagenesis Kit Troubleshooting & FAQs

Problem	Possible Solution
Plasmids cannot be amplified.	Primer design is wrong: re-check the primer design.
	The amplification reaction mixture is not correctly prepared: repeat experiment.
	The amplification reaction is not optimized: the concentration of Mg2+, the amount of enzyme and the amplification program can be optimized.
	The quality of template is not good: long-term storage, repeated freezing and thawing can cause the breakage, open-loop or degradation of the plasmids. Please use freshly prepared plasmids as templates.
There are no or few colonies on the plate.	The efficiency of the competent cell is very low. Use freshly prepared competent cells or competent cells stored properly and make sure the transformation efficiency of competent cells is more than 10⁷ cfu/μg.
	The amount of DNA or ratio of fragments is suboptimal in the recombination reaction. Add the amount of DNA as recommended. Check the concentration of DpnI treated product. DNA concentration must be measured agarose gel electrophoresis and not by any other method such as an absorbance assay.
	The DNA in the recombination cyclization contains impurities inhibiting the reaction; or the volume of unpurified DpnI treated product is more than one fifth of total volume. Perform gel extraction of DpnI treated products. Try to avoid complexing agent (e.g. EDTA) in the recombination reaction. Therefore, we recommend that the purified DNA should be dissolved in ddH2O of pH 8.0 instead of TE buffer.
	Addition of too much DNA to the competent cells: the volume of DNA should not exceed 1/10 the volume of competent cells, otherwise it will reduce the transformation efficiency.
	The transformation inhibitory effect occurs: High concentration of input DNA can inhibit the transformation. In this case, one fifth of the DNA should be used for transformation.
Incorrect site-directed mutation	The primers are not designed correctly. Check the primer design.
	The template plasmids are not methylated. DpnI can only recognize the methylated DNA. Purify the template plasmids from the host strains with functional methylases.
	Too much plasmid used as template. For most plasmids, 1 ng of DNA is enough template for the PCR reaction. Too much plasmid will lead to incomplete digestion by DpnI, which reduces the successful rate of mutation introduction.
Mutations at non-target site.	The template plasmid carries some unknown mutations: confirm the sequence of the template.
	Too many number of amplification cycles: to prevent non-target mutations during the amplification, the number of amplification cycle should not exceed 30 when the amplification efficiency is good.

Site Directed Mutagenesis Kit - Special Remarks

a) When choose the reverse complementary region of primers, please avoid the repetitive sequences. When GC content is 40% to 60%, cyclization recombination efficiency is maximized. If the GC content is higher than 70% or less than 30%, the cyclization efficiency will be significantly inhibited.
b) The double-base mutation strategy can also be used for single base mutations (one of the two sites doesn’t undergo base modification). Therefore, if the amplification cannot be carried out in single base mutation, try to use the double base mutation strategy.