Site-directed mutagenesis

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also known as site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for a variety of applications, but is most commonly used in the study of protein function.

History

The method of site-directed mutagenesis was first developed in the late 1970s. The original method used single-stranded DNA as a template for the synthesis of a complementary strand containing the desired mutation. This method, however, was time-consuming and required large amounts of DNA. In the 1980s, a more efficient method was developed using polymerase chain reaction (PCR), which is now the most commonly used method for site-directed mutagenesis.

Method

Site-directed mutagenesis involves the following steps:

1. Design of a primer that carries the desired mutation.
2. Amplification of the gene of interest using PCR.
3. Transformation of the PCR product into suitable bacteria.
4. Selection of bacteria that carry the mutated gene.
5. Verification of the mutation by DNA sequencing.

Applications

Site-directed mutagenesis is used in a variety of applications, including:

1. Studying the function of specific proteins.
2. Creating proteins with improved properties.
3. Creating proteins with novel functions.
4. Studying the effects of specific mutations on protein function.

See also

• Molecular biology
• Genetic engineering
• Protein engineering

References

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  Site-directed mutagenesis library cloning steps
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  Site saturation mutagenesis