In DNA cloning, mutagenesis allows ones to introduce specific or random changes into a cloned DNA fragment, creating a variety of the gene for further study.
1. Site-directed Mutagenesis:
This method targets a specific location within the DNA sequence for a precise alteration. It's ideal when you want to introduce a well-defined change, like replacing a single amino acid in the encoded protein.
- PCR with mutagenic primers: Primers used for PCR amplification can be specifically designed to incorporate the desired mutation at a specific location during DNA synthesis.
- Restriction enzyme digestion and ligation: The cloned DNA fragment can be cut with restriction enzymes to create a linear piece. Synthetic oligonucleotides containing the desired mutation are then inserted using ligase enzymes to replace the original sequence.
- Cas9-based methods: This approach utilizes the CRISPR-Cas9 system for precise genome editing. A guide RNA directs Cas9 nuclease to a specific location in the DNA, and a repair template containing the desired mutation is introduced for cellular repair machinery to incorporate the change.
2. Random Mutagenesis:
This method performs random mutagenesis throughout the cloned DNA fragment. It is useful for generating libraries of variants with different changes to allow selection of clones with desired characteristics.
- Physical or chemical treatment: DNA is exposed to chemical or physical treatment to induce random mutations. This treatment may use oxidizing agents, alkylating agents, or methods such as UV and X-rays.
- PCR-based mutagenesis: Mutations are induced by amplifying DNA using special PCR conditions that allow specific mutations to occur. By using a primer containing mixed bases, it is possible to induce random mutation only in specific regions.
Bioneer provides various types of mutagenesis services. Base substitution, deletion, or insertion of a specific region is possible. Additionally, mutagenesis services are available that allow tagging a reporter such as GFP to a specific gene and creating a fusion protein by linking multiple genes.
