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Analysis of Mutations that Influence Pre-mRNA Splicing

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405
A rapidly increasing number of human diseases are now recognized as being caused by the selection of wrong splice sites. In most cases, these changes in alternative splice site selection are due to single nucleotide exchanges in splicing regulatory elements. This chapter describes the use of bioinformatics tools to predict the influence of a mutation on alternative pre-mRNA splicing and the experimental testing of these predictions. The bioinformatic analysis determines the influence of a mutation on splicing enhancers and silencers, splice sites and RNA secondary structures. This approach generates hypotheses that are tested using splicing reporter constructs, which are then analyzed in transfection assays. We describe a recombination-based system that allows for the generation of splicing reporter constructs in the first week and their subsequent analysis in the second week.
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