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Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis

Terry H. Landowski, Ibrahim Buyuksal, and William S. Dalton H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa FL 33612


Introduction
Identifying of mutations within specific genes has become an important strategy in determining the diagnosis and prognosis for many diseases, including cancer. Several techniques have been developed to examine heterogeneous tissue samples for specific mutations, including restriction fragment length polymorphism (RFLP), single stranded conformation polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE), among others. These techniques are useful for rapid screening of heterogeneous tissues, and each has its individual strength.1 Using RT-PCR and DGGE, we have examined the cytoplasmic region of the Fas antigen known as the death domain in tumor specimens from patients with lymphoma of various histiocytic origins. These mutations may contribute to the failure of the tumor cells to undergo apoptosis, and thus contribute to the pathogenesis and progression of the disease.2


Materials and Methods
Tumor biopsies were obtained during postoperative resection, and peripheral blood lymphocytes for controls were obtained from normal volunteers. Total RNA was isolated from cryopreserved specimens and 50-100 ng of RNA was reverse transcribed by oligo dT priming. PCR primers for the FasIII region were as previously described2 with the addition of a 42 bp GC clamp on the forward primer.3 Following 35 cycles of amplification in a 9600 Perkin Elmer thermocycler, the PCR products were denatured for 5 minutes at 95
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