The DNA is isolated by phenol-chloroform extraction. Using PCR. target DNA sequences are selectively amplified through repeated cycles of denaturation. annealing with oligonucleotide primers complementary to flanking regions of the target sequence, and primer extension with DNA polymerase (Tag polymerase). The DNA from tumors for ras exon mutation will be screened by single stranded conformation polymorphism (SSCPO. Stands with mutations will appear different from normal strands even if there is only one point mutation. Mutation frequencies are determined by restriction fragment length polymorphism (RFLP) analysis. Amplified DNA (i .e. . ras exons) are directly sequenced using a modification of the dideoxy method to characterize the activation mutations in the ras genes detected in tumors.
