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Using Fluorescence Analysis to Improve Throughput in Human Identity Applications

Hitachi’s FMBIO^® II MultiView is a laser-based, fluorescent image scanning unit ideal for high-throughput DNA typing. The scanning unit features a 20 mw solid-state YAG laser, two photomultiplier tubes, and a dynamic range of up to four orders of magnitude. The scanning unit comes with a Macintosh compatible computer and the following Hitachi Software which: 1) the ReadImage Program, which controls the scanning unit as it generates digital images of gels, 2) the FMBIO^® Analysis Program, which permits viewing and analysis of a scanned image containing one or more fluorophores, and 3) the new STaR CallÔ Software, which automatically converts base values to alleles and facilitates rapid transfer of genetic data to the Combined DNA Index System (CODIS). We will describe how the instrument works and present an introduction to multicolor fluorescence imaging on the FMBIO^® using the dyes fluorescein, carboxy-tetramethylrhodamine, and carboxy -X- rhodamine, which are used in Promega’s GenePrintÔ PowerPlexÔ STR System.

We will also describe the development of high throughput Short Tandem Repeat (STR) DNA analysis using the powerful combination of the PowerPlexÔ STR System from Promega, Hitachi’s R³Ô Pre-cast Gels, the FMBIO II^® Multiview System, and STaR CallÔ genotyping software. This combination greatly improves throughput and accuracy over traditional silver staining and one-color fluorescence systems. Pre-cast gels decrease preparation time by eliminating the need to pour acrylamide gels and wait for polymerization. Use of internal size markers allows additional samples to be run on each gel by reducing the need to position allelic ladders next to each DNA sample. Internal size markers also improve precision by eliminating inaccuracies from gel smiling or frowning. Using the three-color imaging capabilities of the FMBIO II^® to analyze the Promega PowerPlexÔ-generated PCR products, eight STR loci can be analyzed from a single PCR amplified sample.

Hitachi’s new STaR CallÔ genotyping software was developed for high throughput analysis and rapid entry of genotypes into the CODIS PCR/STR database. Consistent sizing and reproducibility in allele assignments from gel to gel and operator to operator have been demonstrated using STaR CallÔ.

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