In this video - Site-Directed Mutagenesis Explained | Understanding the Basics - I look at how you can mutate DNA in the lab.
Site-directed mutagenesis is a powerful technique in molecular biology that can introduce specific mutations into a DNA sequence. The method has been instrumental in understanding how proteins function and how genes are regulated.
The Process of Site-Directed Mutagenesis
Site-directed mutagenesis involves several key steps:
- Preparation of DNA: The DNA sequence of interest is either cloned into a plasmid or selected from a plasmid library.
- Designing the Primer: A primer is designed to bind specifically to the region of DNA where the mutation is desired. This primer is about 20 bases long, with one crucial difference—it contains the new base that will introduce the mutation. More information on Primer Design.
- Creating a Single-Stranded DNA Template: The double-stranded plasmid DNA is converted into a single-stranded form. This single strand serves as the template for the primer to bind.
- DNA Synthesis and Ligation: Once the primer is bound, DNA polymerase synthesises the complementary strand, incorporating the new base. DNA ligase is then added to seal the DNA strand, completing the synthesis.
- Introduction into Bacteria: The newly mutated plasmid is introduced into bacteria, where the bacteria's natural DNA repair mechanisms take over. These mechanisms either repair the mismatched DNA back to its original form or incorporate the new mutation.
Applications of Site-Directed Mutagenesis
The ability to change a single base in a DNA sequence provides scientists with a powerful tool to explore how specific mutations affect protein function and gene expression. This technique has enabled numerous studies, allowing researchers to pinpoint individual amino acids' roles in proteins and dissect complex genetic regulatory networks.
From Site-Directed Mutagenesis to CRISPR
While site-directed mutagenesis has been a common lab technique for decades, the advent of CRISPR technology has revolutionised the field of genetic engineering. CRISPR offers a more efficient and precise method for making targeted genetic changes, but the foundational principles of mutagenesis laid the groundwork for these modern advances.
