Two ways of detecting point mutations - temperature gradient capillary electrophoresis and free solution cze in acidic, isoelectric buffers

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Project objective

Research objectives and content Temperature Gradient Capillary Electrophoresis (TGCE) is a newly developed methodology which has shown to be a promising method for detecting point mutations. This approach combines the principal of Temperature (Denaturing) Gradient Gel Electrophoresis (TGGE/DGGE), widely used methods for identifying sequence variants, and capillary zone electrophoresis (CZE) which represents the most advanced electrophoretic technique. Because of the advantages of CZE over slab gels, we believe that TGCE has a potential of becoming a privileged tool for mass screening of genetic defects in humans. I propose to focus on the optimization and improvement of the newly developed TGCE device in terms of efficiency and accuracy. This implies as well the search for new separation media exhibiting low viscosity. Secondly I propose to explore a totally new approach, which consists of separating two or more oligonucleotides of identical size but differing in one base at a given point in their sequence. Choosing the right pH conditions these equally long oligonucleotides can be separated because of charge difference and without adding any sieving medium. Training content (objective, benefit and expected impact) The host laboratory has been very active in the development and validation of CZE for diagnostic purposes. The stay in Prof. Righetti's laboratory, will complement my present fundamental training and is parallel to my final aim, which is to apply my knowledge, competence and force to public health and the cure of diseases. Links with industry / industrial relevance (22) The TGCE mode can be easily adapted to multicapillary CZE device. Such instrumentation, coupled with individual voltage control on each channel in the array would allow for running various thermal gradients at a time. This approach would increase the efficiency of TGCE enormously and could become a formidable tool for large-scale screening of single nucleotide variations in genomic DNA.

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Participants

• UNIVERSITA DEGLI STUDI DI VERONA · VERONACoordinatorItaly

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