CAPILLARY ELECTROPHORESIS METHODOLOGY FOR IDENTIFICATION OF CANCER-RELATED GENE-EXPRESSION PATTERNS OF FLUORESCENT DIFFERENTIAL DISPLAY POLYMERASE CHAIN-REACTION

The mRNA differential display technique is a method for molecular surv ey and analysis of differential gene expression in eukaryotic cells an d tissues. We have previously described the use of ABI Prism fluoresce nt technology to specifically amplify expressed sequence tags (ESTs) f rom several different biological paradigms. High throughput, fluoresce nt differential display performed on an automated sequencer (ABI 377) has proven to have significant cost cutting and time saving attributes compared to that of the radioactive differential display. Additionall y, fluorescent tagged mRNA specific reverse transcription and PCR decr eases the number of the inherent artifacts associated with radioactive differential display. We report hen the application of a capillary el ectrophoresis system (ABI 310) to the identification of fluorescent di fferential display generated EST patterns. RNA samples from human and animal breast cancer paradigms were exposed to this technique and anal yzed by the ABI 310 and the ABI 377. GeneScan and Genotyper software a pplications were used for rapid and semi-automated characterization of fluorescently labeled ESTs. Automated sample loading and uniform samp le electrophoresis are among the main advantages of this system which significantly increase the precision and reproducibility of fluorescen t differential display.