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

ut the wild type cDNA. Patient samples that demonstrated heteroduplex formation on DGGE analysis were incubated overnight at 37 C with 0.5U DraI, denatured for 5 minutes at 95 C, and allowed to reanneal at 55 C for 1 hour. Digested products were loaded on the DGGE acrylamide gel, and electrophoresed 3.5 hours under conditions described above (Figure 3). This treatment resulted in the disappearance of one of the heteroduplex bands in specimens with the polymorphism and the appearance of a truncated homoduplex band (see lanes 1, 4, 5, 8, and 9 of Figure 3). Patient specimens that are homozygous for the polymorphism display only the truncated homoduplex (lane 10), while patient specimens with mutations in addition to the polymorphism retained two heteroduplex bands (lanes 7 and 11).


Conclusions

Crosslinking of the Fas antigen by antibody or specific ligand induces apoptosis in susceptible cells. Mutations within the signal transducing domain may render the cells resistant to programmed cell death, and contribute to the progression of neoplastic disease. We have utilized the methods of RT-PCR and DGGE to detect small changes in the coding sequence of the Fas antigen in hematological tumors.

In contrast to many other rapid screening techniques, DGGE is based on known physical parameters of a particular gene. By calculating the predicted melting temperature of a segment of DNA, analytical conditions can be optimized for maximum accuracy and sensitivity.5 We have demonstrated that DGGE can be used to detect as little as 1% of total RNA containing a single base change with non-radioisotopic techniques. In addition, with a combinati
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