Analysis of site-directed mutagenesis constructs by capillary electrophoresis using linear polymer sieving matrices

Site-directed mutagenesis is a novel molecular biology tool, which introduces mutations into DNA fragments of interest in a well-defined manner. Sequences with designed mutations can be generated in this way to express altered protein sequences for structure-function relationship studies. However, prior to gene expression, it is important to analyze the DNA construct to see whether the introduction of the mutation was indeed successful. Currently DNA sequencing is the method of choice for this verification. This paper introduces the combination of primer extension and capillary electrophoresis using linear polymer sieving matrices as an efficient alternative for this type of mutation analysis. The site-directed mutagenesis construct served as template in the primer extension reaction that employed a fluorophore labeled primer in close proximity to the mutation. Appropriate ddNTP was used to block the extension when the mutation was present, while the other three dNTPs enabled elongation of the primer. Alternatively, non-labeled primers can be used with the proper fluorophore labeled ddNTPs to block the reaction. Rapid analysis of the labeled primer extension products (mutant or wild type) was obtained by capillary electrophoresis using denaturing sieving matrix and laser-induced fluorescence detection.