Part 2: Non-PCR-Based Site-Directed Mutagenesis | Cassette Mutagenesis скачать в хорошем качестве

Part 2: Non-PCR-Based Site-Directed Mutagenesis | Cassette Mutagenesis

‪@BiologyWithChandra‬    • Part 1: Site-Directed Mutagenesis | Introd...   #SiteDirectedMutagenesis #MolecularBiology #GeneticEngineering #ProteinEngineering #DNAMutation #InVitroMutagenesis #PCR #PCRBasedMutagenesis #CassetteMutagenesis #PrimerExtension #MutagenicOligonucleotides #GeneRegulation #WildTypeVsMutant #AminoAcidSubstitution #Biotechnology #LifeSciences #GeneticsLecture In this video, we explain Cassette Mutagenesis, a non-PCR-based method of Site-Directed Mutagenesis used to introduce specific mutations into cloned DNA. Cassette mutagenesis involves replacing a restriction fragment from the DNA of interest with another fragment that contains the desired mutated sequence. This method depends on the presence of two restriction enzyme recognition sites flanking the region to be mutated. With the help of restriction enzymes, the target region in the wild-type gene is removed. A synthetic double-stranded oligonucleotide cassette containing the desired mutation is then inserted in place of the removed fragment and ligated into the vector. The gene present in a suitable vector is cleaved with two restriction enzymes, releasing a small DNA segment. Since the enzymes cut at sites flanking the region of interest, only the desired portion is replaced. The synthetic duplex DNA cassette is then ligated into position. This method: Allows relatively large-scale changes Can introduce single or multiple mutations Has very high efficiency Is simple and straightforward However, a major disadvantage is that it requires unique restriction sites flanking the region of interest. This lecture is helpful for students of Molecular Biology, Biotechnology, Genetics, and Life Sciences preparing for competitive exams like CSIR-NET and GATE.