Gabriel E. Novick, M.D., Ph.D., Sandra M. Sovinski, B.S. and Mohammed A. Tahir, Ph.D.
Indianapolis-Marion County Forensic Services Agency, 40 South Alabama Street, Indianapolis, Indiana, 46204

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RFLP analysis is a well established and very informative test for the forensic identification of body fluids and tissues. The advent of PCR-based typing methods (DQa, Polymarker, and now STR analysis), plus some disadvantages of the procedure have made RFLP analysis a less appealing approach, especially for new labs. Compared to PCR analysis, radioactive RFLP typing entails the cost and safety considerations of working with radioactivity, the limitations in both quantity and quality of DNA sample, and the time required to complete the analysis. Non-isotopic detection of RFLP patterns has extended the life of the technique by eliminating the risks, reducing the costs and decreasing the analysis time.

We have optimized a protocol, a slightly modified version of the FBI procedure, for chemiluminescent detection of RFLP profiles with high sensitivity and consistent low or non-existing background.

Our validation studies included the following: (1) ladder evaluation; (2) sensitivity; (3) environmental insults, contamination and matrix; (4) population studies, including K562 data analysis; (5) simulated casework; (6) non-probative casework; (7) and proficiency testing.

We are currently typing six loci: D2S44, D10S28, D17S79, D5S110, D4S139 and D1S7. The minimum sample that can be detected is 5 ng of K562 DNA, 5 µl of blood and 2 µl of semen, with a maximum throughput per analyst of 24 membranes processed with probes in 13 days (60 films/week/analyst).