With the support of our advanced protein engineering platform established for many years, Creative BioMart has developed a variety of site-directed mutagenesis methods. Our scientists provide you with a comprehensive customized site-directed mutagenesis library construction service.

Site-directed mutagenesis is a method to produce specific and targeted changes in double-stranded plasmid DNA, which can change the amino acid composition, destroy the binding site of transcription factors, or produce fusion proteins. It uses custom-designed oligonucleotide primers to confer the desired mutations in a double-stranded DNA plasmid. Site-directed mutagenesis includes changing restriction sites in the vector, inserting or deleting vector elements, adding/removing tags, base substitution, sequence deletion/insertion, and promoter insertion/exchange.

Site-directed mutagenesis is a highly versatile technique. By using overlapping and complementary oligonucleotide pairs to perform a simple PCR reaction, almost any desired mutation can be introduced into the gene carried by the plasmid. Site-specific mutagenesis is an indispensable tool for structure and function research.

Fig 1. Schematic representation of site-directed mutagenesis strategy. (Allemandou F, et al., 2003)

Methods of Site-Directed Mutagenesis

• Kunkel's method: inserting the DNA fragment to be mutated into the phagemid, and then transform it into an E. coli strain lacking dUTPase and uracil deglycosidase. A technique was introduced to reduce the need to select mutants.
• Cassette mutagenesis: in this method, a DNA fragment is synthesized and then inserted into a plasmid. This method can generate mutants with close to 100% efficiency, but is limited by the availability of suitable restriction sites flanking the site to be mutated.
• PCR site-directed mutagenesis: the use of polymerase chain reaction and oligonucleotide "primers" can overcome the restriction of restriction sites in cassette mutagenesis, which can generate larger fragments covering two convenient restriction sites.
• Whole plasmid mutagenesis: this technique can create plasmid mutagenesis libraries, ranging from single mutations to comprehensive codon mutagenesis of entire genes.

Applications of Site-Directed Mutagenesis

• Studying on the structure of protein functional sites.
• Enzyme activity optimization.
• DNA component function or component interaction.
• Gene therapy.

Service Principle

Creative BioMart is based on innovative, pragmatic and honest, adhering to the tenet of "Quality is our life, providing customers with the best quality service", providing customized services to customers around the world.

We will be glad to discuss details of intended interaction studies with you and develop experimental strategies/methods tailored to your requirement. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, Monday to Friday. Please do not hesitate to contact us for more information or to discuss in detail, if you are interested in our services.

References

1. Bachman J. (2013) Site-directed mutagenesis. Methods Enzymol. 529, 241-8.
2. Costa GL, Bauer JC, et al. (1996) Site-directed mutagenesis using a rapid PCR-based method. Methods Mol Biol. 57, 239-48.
3. Allemandou F, Nussberger J, Brunner H R, et al. (2003) Rapid site-directed mutagenesis using two-PCR-generated DNA fragments reproducing the plasmid template[J]. Journal of Biomedicine and Biotechnology. 2003(3): 202-207.

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