4 - Molecular Analysis of Genetic and Epigenetic Alterations in Human Cells

Abstract

Publisher Summary Recombinant DNA technology and DNA sequencing techniques allow mutations induced in mammalian cells to be analyzed at the molecular level. If the gene in which mutations are induced has been cloned, gross structural alterations are readily detected by Southern analysis. Point mutations or small deletions are much more difficult to analyze. Several different mutation systems have been subjected to molecular analysis. Mutations in endogenous genes are analysed by the cloning and sequencing of the entire mutated gene from the mammalian genome. Endogenous genes are the ideal system to use. Mutations in mammalian viral genes are analyzed by the recovery of the mutated viral particles and the sequencing of their DNA. Shuttle vectors are designed in which a target gene is inserted into a plasmid that is capable of replicating both in bacterial and mammalian cells. The plasmid is mutated inside the mammalian cell, after which it is recovered from the mammalian cell and used to transform suitable indicator bacteria in which mutations induced in the target gene inside the mammalian cell is easily identified and analysed in the bacteria.