Patrick Williams, Sue Seim, Linda Wiessner, Tami Maus and Kathy Graham
Memorial Blood Center of Minnesota, Minneapolis, MN

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Analysis of Restriction Fragment Length Polymorphism (RFLP) for paternity testing was initially developed using radioisotope labeled locus specific probes. While sensitive, this technique has proven to be time consuming and expensive to perform. Fluorescent and chemiluminescent probe detection chemistries have subsequently been developed as alternatives to radioisotope methods. However, these chemistries can also require long incubation times and costly detection equipment for data collection. Colorimetric chemistries, such as NBT/BCIP substrate used in conjunction with Alkaline Phosphate probes have also been used to visualize RFLP patterns. Such chemistries offer several advantages over other systems; increased resolution of bands, shorter development times as compared to isotopes, and no requirements for expensive equipment, film development labs or film. A disadvantage of colorimetric systems has been the need for larger amounts of DNA usually 1.0 ug or greater). Recent advances in the application of Charge Coupled Device (CCD) video systems and digital imaging to DNA testing have made it possible to use these systems to record the image of an RFLP Southern blotted membrane and then process the collected data. Stained membranes were imaged with an Alpha Innotech IS-2500 Digital Imaging System using 1/30^th second exposures and digitally enhanced using both high pass and contour digital filtering algorithms. Image analysis was performed using DNA Pro Scan PRO-RFLP software. This system has been demonstrated to provide high resolution imaging of colorimetric stained RFLP membranes and rapid data analysis. Versatility of the system allows for future developments in the area of increased staining sensitivity used in conjunction with smaller sample size.