Forensic application of rapid DNA preparation for muliplex PCR

9TH INTERNATIONAL/6TH EUROPEAN JOINT SYMPOSIUM ON PURINE AND PYRIMIDINE METABOLISM IN MAN cellular degradation of apoB and its translocation into the endoplasmic reticulum were also investigated. Results ApoB secretion was decreased four fold in CsA-treated HepG2 cells compared to control cells. Using a semi-permeable HepG2 degradation assay, the presence of CsA resulted in a 36.7% increase in the degradation of apoB. The translocation of apoB in the presence of CsA was by decreased by 37% based on results obtained using a recently developed translocation protocol. Conclusion We suggest that the hepatic production of apoBcontaining lipoproteins is decreased in the presence of CsA, and that this decrease can be attributed to both an increase in the degradation of apoB and a decrease in the percentage of apoB translocated into the endoplasmic reticulum. CsA was kindly provided by Dr. Randall W. Yatscoff, University of Alberta.

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FORENSIC APPLICATION OF RAPID D N A PREPARATION FOR MULIPLEX PCR Bourgoin, S., Mailly, F., Sarafian, V., Bergeron, J., and Linard, C., Lab. Sc. Judiciaires M~d. L~gale, 1701 Parthenais Montreal, Qc, H2L 4K6 and UQTR, D~pt. ehimie biologie, C.P. 500, Trois-Rivi~res, Qc, G9A 5H7, Canada

Simultaneous amplification of Short Tandem Repeats loci is increasingly employed in forensics to generate discriminating genetic profiles. However, laborious organic methods for DNA preparation still hinder speedy sample processing. Objectives Optimization of rapid, cost-effective DNA preparation protocols was undertaken for a variety of small forensic-like samples based on published methods (Cancer cells 1989; 7: 209-14, Hum. Mol. Genet. 1993; 2: 159-63). The aim was to obtain genetic material amenable to multiplex amplification and fluorescence detection. Methods DNA was prepared from hair, sperm stains, bloodstains (pre-washed in sucrose buffer), skin and buccal swab samples with known profiles. Samples were either incubated in NP-40/ Tween buffer plus various additives (proteinase K, DTT, NaOH) or subjected to an organic extraction protocol. Yield was quantified by slot blot. Aliquots were subjected to multiplex amplification and detection on an ABD 377 Genescanner. Results For all samples, the optimal 'fast protocol' yield was comparable or superior to that obtained by organic extraction. Bloostains and hairs required proteinase K and boiling with NaOH while only proteinase K was needed for swab samples. Incubation with DTT prior to boiling with NaOH was optimal for sperm samples. Successful amplification and profile analysis were achieved with all optimum preparations with sensitivity similar to organic preparations. Conclusions High quality profiles can be obtained from rapidly prepared DNA in multiplex PCR. This economical approach reduces the risk of sample mixup and may be invaluable in cases requiring an urgent response. A composite protocol is being tested to resolve profiles from sperm/blood mixtures.

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TELOMERASE DISTINGUISHES RENAL CELL CARCINOMA FROM RENAL ONCOCYTOMA Bhuiyan, J., Roberts, S. G., Thibodeau, S.N. and O'Kane, D.J., Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, Minnesota 55905.

Cellular immortalization is concomittant with expression of telomerase, an immortalizing enzyme that utilizes an integral RNA template to extend telomeric DNA with d(TTAGC~)n tandem repeats. Telomerase is absent from most benign and normal cells but is expressed in most cancerous cells making it a useful 380

surrogate tumor marker. Renal cell carcinoma (RCC) is the most common neoplasm in the adult kidney, the only effective therapy being complete surgical resection including nephrectomy. Renal oncocytoma (ROC) is a benign tumor requiring only observation. RCC and ROC may appear similar radiographically and histologically and differential expression of telomerase would be valuable in such cases in which nephrectomy could be avoided if a benign tumor can be documented. Telomerase activity has been detected in RCC but its status in ROC has not been investigated. Objectives To ascertain the status of telomerase activity in ROC, and to determine whether ROC can be distinguished from RCC based on telomerase activity. Methods Tissue specimens were obtained from patients who underwent either partial or complete nephrectomy for renal masses. Tissue cytosols from ROC, RCC, and normal kidney were assayed for telomerase using the telomere repeat amplification protocol (TRAP) asssay. The telomerase positive cytosols produced a characteristic ladder of bands at 6 base increments starting at 50 nucleotides with an additional band of a 36 base-pair internal standard. Results Telomerase activity was absent in 11/12 (92%) ROC. Low activity was detected in 1 ROC. Telomerase activity was found in 33/35 (94%) RCC specimens that spanned the full spectrum of histologic grade (1-4) and cell type. It was observed that a low grade (1-2) RCC had either low or no activity. The normal kidney tissue (N = 31) lacked telomerase activity. Conclusions Telomerase expression distinguishes RCC from benign ROC. Therefore, its measurement may play a pivotal role in surgical decisions regarding the extent of nephrectomy.

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NOVEL IN VITRO METHODS FOR A S S E S S M E N T OF ENVIRONMENTAL TOXICITY USING A HUMAN CELL-LINE: APPLICATION TO A S S E S S MENT OF ENVIRONMENTAL HEALTH Hasspieler, B.M., Haffner, G.D. and Adeli, K., Dept. of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B 3P4, Canada.

A battery of in vitro testing methods has been developed in our laboratory which utilise the human hepatoma cell line, HepG2, as the cellular target for pollutant-mediated health effects. The four test methods form a comprehensive screening system for a wide variety of toxicants that are prevalent in environmental matrices such as food, air, water, soil and aquatic sediments. Objectives To optimise the four tests for the purpose of highthroughput screening of environmental samples, to calibrate the tests using known standard toxicants that are of relevance to environmental health, and to apply the testing system to a variety of pollutant-laden environmental samples in order to evaluate the human health hazard posed by these samples. Methods The four test methods developed include: (a) a cytotoxicity test based upon uptake of the non-toxic dye, Neutral Red, by viable cells and reduced uptake by cells subjected to toxic injury, (b) a DNA strand breakage test based upon an alkaline unwinding assay, followed by hydroxylapatite DNA chromatography, which quantifies the single-strand breakage mediated by toxicants causing physical damage to the strand, (c) a DNA repair assay based upon incorporation of ~H-thymidine into cells that undergo repair of damaged DNA via unscheduled DNA synthesis, and (d) an enzyme induction assay based upon increased levels of the cytochrome P-450 IA1 enzyme subfamily as indicated by ethoxyresorufin O-deethylase. Results The four tests were shown to be responsive to standard toxicants such as heavy metals, polycyclic aromatic hydrocarbons, PCBs, dioxins, nitroaromatics and quinones. The tests were also responsive to toxicants occurring in various environmental CLINICAL BIOCHEMISTRY, VOLUME 30, JUNE 1997