( C and reannealed for 1 hour at 55 C to...)Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis

Melting temperature of the 430 bp PCR product was predicted using MacMelt software, and confirmed by analysis on a 6% acrylamide gel (37.5:1) with a perpendicular gradient of 12.537.5% denaturant. Electrophoresis in the DCode system was at a constant temperature of 60 C in 1x TAE buffer. Gels were run at 100 V for 2.5 hours. Optimum strand separation occurred at 2324% denaturant, which correlated to the predicted melting temperature of 6570 C. All further analyses were done in a parallel gradient of 1530% denaturant at 60 C, with running time extended to 4.5 hours. PCR products were detected by ethidium bromide staining.

Results and Discussion
Hematological tumors typically contain a heterogeneous population of cells that includes both neoplastic and normal cells. The identification of tumor specific mutations requires a method that can separate mutant sequences from normal sequences for isolation and identification. Sensitivity of the techniques is important in diseases such as lymphoma, which typically contain large numbers of normal stromal cells and infiltrating lymphocytes.

To determine the sensitivity of the DGGE analysis, we took advantage of a previously identified polymorphism in the Fas antigen, in which bp836 is changed from C to T.⁴ Direct sequencing of PCR products has shown that the T cell leukemia cell line CEM is heterozygous for the alternative codon, ACT, while the myeloma cell line 8226 expresses only the higher frequency codon, ACC. RNA was isolated from CEM and 8226 cell lines, and titrated at 1%, 5%, 10%, 30%, and 50% to determine the minima
'"/>

Source: