Separation, real-time migration monitoring and selective zone retrieval using a computer controlled system for automated analysis.
High throughput routine analysis of dsDNA fragments or molecular weight determination of proteins via electrophoresis still require significant efforts to obtain results of high reproducibility and accuracy. This paper analyses the use of a fully automated multi-purpose real-time gel electrophoresis system in these applications and evaluates the benefits of this new concept for routine and research. By comparing currently used systems with this new approach, it also addresses the analytical use of information resulting from real-time dynamic migration monitoring via fluorescence photometry over commonly obtained results from post-run fixation, visualization and evaluation at a single time of the separation. The simultaneous separation of components in multi-gel systems, pre-concentration of sample components, and the ability to perform in-gel assays for biological activity are discussed on basis of routine gel separations of restriction enzyme digested DNA fragments, native and denaturing protein separations and enzyme activity determination. Interactive, selective retrieval of separated components in the nano- to microgram range is carried out for real-time isolation of proteins or dsDNA fragments. Results are compared to blotting in respect to ease of use and transfer efficiency and for immediate availability of macromolecules for sequencing or mass spectroscopy. The Windows-based operating software is critically reviewed for functionality, user-friendliness, graphical data representation and GLP compliance for LIMS oriented forensic or certified laboratories. A statistical evaluation of lane-to-lane and gel-to-gel reproducibility of mobility data, quantification and molecular weight determination concludes the paper.
Gombocz
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LabIntelligence, Inc., Menlo Park, California, USA.
Cortez
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D001322:Autoanalysis / Q000379:methods*; D003201:Computers; D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D004247:DNA