Nibedita Mahanti, Kathy Kuebler, Dorothy Martus, Heather Spillane, Lynne Helton, Julie
Howenstine, Stephen Milligan and Charles Barna
Michigan State Police, 714 South Harrison, East Lansing, MI 48823
Conventional 32P detection of RFLP is widely being used for the forensic casework application despite its limitations. The long exposure times, waste disposal difficulties and the health hazard concerns have made this procedure less attractive to the forensic DNA caseworkers. Chemiluminescent detection using alkaline phosphatase labeled oligonucleotide probes and Lumi-phos plus™ as a substrate for non-radioactive detection of RFLP patterns offers a much faster exposure time with equivalent or greater sensitivity when compared to 32P detection. This dramatically reduces the turnover times for probes. We have used D1S7, D2S44, D4S139, D5S110 and D17S79 as probes against DNA extracted from liquid/stain blood samples and were able to develop RFLP patterns within hours as compared to days using radioactive probes. In our hands, chemiluminescent probes provide a lower detection limit of 25ng of DNA and optimum of 100-200ng. Early reports using chemiluminescent probes often indicated the lack of sensitivity (allele dependent) within 1-2 kb range. A group of selected human DNA samples having alleles in this range was evaluated. Results of this study revealed that alleles falling into this range could be reliably detected. Based on our observation, chemiluminescent probes coupled with Lumi-phos plus™ detection system provides equivalent or greater sensitivity when compared to 32P detection. The exposure periods for Lumi-phos never exceeded five hours as opposed to exposures for 32P normally taking several days. Due to the lack of availability of some of the VNTR probes for chemiluminescence, we investigated the possibility of probing the membranes with 32P probes in the middle of the chemiluminescent probing cycle. These interchanges did not affect the results of either chemiluminescent or 32P probing. Based on these observations, chemiluminescent detection looks promising as an alternative method to 32P detection for RFLP analysis.
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