( l amount of polymorphic cDNA ...)Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis

Tumor specimens from patients with lymphoma were examined for mutations in the signal transducing domain of the Fas antigen by RT-PCR and DGGE analysis. Figure 2 shows a representative ethidium stained DGGE gel including 10 patient samples. The CEM T cell line serves as a positive control, and demonstrates the heteroduplex bands formed by polymorphic and wild type cDNA. The myeloma cell line 8226, which serves as a negative control, has been fully sequenced and shown to be wild type sequence. Of the patient specimens demonstrated in Figure 2, two display a single band characteristic of wild type sequence. Patient specimens 4, 5, 7, 9, and 10 demonstrate heteroduplex band formation, indicating an alteration in the cDNA sequence. These bands were extracted from the gel and reamplified by PCR for direct sequencing. In addition, the altered mobility of the single band seen in patient 8 is suggestive of a homozygous mutation. This band was also extracted for sequence analysis.

With the high frequency of polymorphisms reported at bp 836 in the Fas antigen, we sought to develop a screening method to distinguish the known polymorphism from other unknown mutations that could have functional significance. Expression of the alternative codon, ACC, introduces a unique restriction site for the enzyme DraI which is not present in the wild type cDNA sequence. Cleavage of the FasIII cDNAs with DraI results in an 368 bp fragment when the polymorphic codon is expressed, while this enzyme does not c
'"/>

Source: