Molecular Cloning and Reverse Genetics.

Eugenia S Bardossy

doi:10.1007/978-1-0716-5496-5_8

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Molecular Cloning and Reverse Genetics.

Eugenia S Bardossy

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Abstract

This chapter describes a detailed molecular biology protocol for introducing specific point mutations into the chikungunya virus (CHIKV) genome using a reverse genetics strategy. The method utilizes an overlapping PCR-based approach to generate a mutated DNA fragment, which is then cloned into a pre-engineered CHIKV infectious clone plasmid. The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector. It also includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing to confirm the mutation, and plasmid purification via miniprep. The document is structured with a clear introduction, a list of all required reagents and equipment, and a step-by-step methods section.

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Cloning, Molecular Reverse Genetics Polymerase Chain Reaction Chikungunya virus Genome, Viral Plasmids Mutagenesis, Site-Directed Animals Genetic Vectors Point Mutation Humans Molecular cloning Overlapping PCR Reverse genetics Site-directed mutagenesis Journal Article

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Eugenia S Bardossy. 2027. Molecular Cloning and Reverse Genetics.. https://doi.org/10.1007/978-1-0716-5496-5_8

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