Jennifer M. Worley, Peter T. McNamara, and Elaine S. Mansfield
Molecular Dynamics, 928 East Arques Avenue, Sunnyvale, CA 94086

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Fluorescent detection methods offer safe, sensitive alternatives to radioactivity. The unique strength of fluorescent detection, however, lies in the use of multiple colors of fluorescent tags to increase productivity. Multi-color assays may be used to increase sample throughput, to enhance the informativeness of an assay, or allow co-analysis of several molecules, including the use of an internal standard to normalize results.

The FluorImager™ System (Molecular Dynamics, Sunnyvale, CA) is a general purpose fluorescence scanner that allows detection and analysis of a wide variety of sample formats and fluorescent labeling methods. We have enhanced the instrument’s sensitivity for improved detection of a wide range of fluorescent molecules, without sacrificing the instrument’s versatility. The Fluorochrome Separation software utility allows efficient separation of fluorescent emission spectra, without band drop-out in co-migrating bands. This system provides typing of microsatellite and short tandem repeat (STR) loci. We scanned samples of the new GenePrint™ STR systems (Promega, Madison, WI) labeled with two fluorescent dyes, and analyzed it using Fragment Analysis software. This software allows accurate sizing of unknown fragments in one- and two-color gel applications. Alleles are then called automatically using the Genetic Typing software module. We have also tested the compatibility of the microplate-based TaqMan™ (Perkin-Elmer, Norwalk, CT) assay. This assay uses an internal fluorogenic probe for single-step quantitative analysis of PCR products when analyzed with our ImageQuant™ analysis software. We have demonstrated analysis of both one-and two-color TaqMan assays scanned on the FluorImager.