David L. Steffens, Ph.D., Reena Roy, Ph.D., John A. Brumbaugh, Ph.D.
Li-Cor, Inc., Biotechnology Division, P.O. Box 4000, Lincoln, NE 68504-5000

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An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR alleles in multiplex reactions. Up to five STR loci and one gender locus were visualized in one lane after amplification of the selected STR and gender alleles in a single PCR reaction (Fig. 1 & 2). Multiplexing was also used to detect at least three gender loci in a single amplification reaction (Fig. 3).

The strategy for IR fluorescence detection of PCR products was to include IR labeled dATP molecules in mixtures of various Promega GenePrint^™ kits during amplification. Allelic ladders were amplified similarly. After a brief procedure to remove unincorporated IR-dATP molecules, the products were loaded on electrophoresis gels. Image data was collected on-line in real time. Allelic patterns were displayed as autoradiogram-like images, scored visually and quantitated by image analysis software. Family studies (Fig. 4), paternity analyses and various simulated forensic case samples were analyzed. The applicability of this technique to rapid human identification and relationships will be demonstrated.