Figure 5.
(A) Detection of a tumor-specific somatic mutation by SSCP and heteroduplex analysis. DNA samples were PCR-amplified by using primers flanking the first coding exon of TRC8. The products were denatured, separated on a nondenaturing MDE gel and detected by silver staining. Samples included matched tumor and normal DNAs from patients 1 and 7 (lanes 5–8, respectively) and an unrelated normal control (AG4103, lane 9). A separate SSCP gel was used to isolate four individual SSCP bands from RCC no. 1 (lane 5, marked by an arrow or arrowheads). The excised bands A-D corresponded to the indicated bands in lane 5 from top to bottom. These last templates were reamplified and analyzed by SSCP to determine whether they contained mutant or wild-type sequences. Comparison to lane 5 suggested that bands A and C contained primarily mutant DNA, band B was a mixture of mutant and wild-type, and band D was wild-type only. Results for SSCP (Upper) and heteroduplex (Lower) are shown. (B) RCC no. 1 contains a 12-bp duplication in the 5′ untranslated region. Purified PCR products shown in A (lanes 1–4) were sequenced. The mutation consisted of a 12-bp direct duplication (underlined) at bp position −73, which was present in the tumor sample but not in the corresponding normal DNA.