PCR-based approaches for detection of mutated ras genes.

T h e ras genes encode 21-kD GTPbinding proteins (p21 ras) that cycle between an inactive GDP-bound form and an active GTP-bound form. The p21 ras proteins are thought to play an important role in signal transduction that eventually controls proliferation and differentiation of many different cells. The ras (pro to -onco)genes can be activated as oncogenes by simple point mutations, which substitute a single amino acid residue at a critical position in the protein product. Mutated ras genes occur with a high frequency in common human cancers, including adenocarcinoma of the lung, colorectal cancer, myeloid leukemia, and adenocarcinoma of the pancreas.O) Originally, the transforming ras mutations were determined by transfection of NIH-3T3 cells, followed by cloning and sequencing of the oncogene. The mutations found are almost without exception point mutations at codons 12, 13, or 61 of the highly homologous ras genes H-ras, Kras, and N-ras. The introduction of the polymerase chain reaction (PCR) technique has greatly simplified the procedure for analyzing tumors for the presence of one of these point mutations. A source for the DNA to be analyzed may be either fresh or frozen tissue samples. Also formalin-fixed, paraffin-embedded tissue sections may be used as starting material, allowing retrospective analysis of archival material. Different methods have been devised to detect the common point mutations in the PCR-amplified ras gene segments, as shown in Table 1. We will discuss these different methods in this review. Allele-specific Oligonucleotide Hybridization Allele-specific oligonucleotide hybridization (ASO) to detect point mutations is based on the fact that an oligonucleotide with one mismatch binds more weakly to its (imperfect) complement than a perfectly matching one. By performing hybridization and washing just below the dissociation temperature (Tm) of the perfectly matching oligonucleotide, one can discriminate between a perfectly matching oligonucleotide and an oligonucleotide with 1bp mismatch. (2) In short, PCR-amplifled DNA is spotted onto a nylon (or nitrocellulose) filter and hybridized to a radioactively labeled oligonucleotide which corresponds to the wild-type sequence of the particular ras codons. The filter is washed under stringent conditions and hybridization is detected by autoradiography. This procedure detects the presence of the wildtype allele of the gene segment. Duplicate filters are then hybridized with different, labeled oligonucleotides, each of which corresponds to one of the expected point mutations in the gene segment. Hybridization of one of these allele-specific (point mutationspecific) oligonucleotides shows the presence of the corresponding point mutation. Hybridization and stringent washing may be performed in the presence of 3 M tetramethylammonium chloride.O) At this concentration the G-C and A-T base pairs have similar stabilities, so that oligonucleotides of the same length have the same T m, regardless of the base composition. This allows hybridization and washing of the filters with different oligonucleotides next to each other in one procedure. Using this method, large series of DNA samples can be analyzed in a short period of time for the presence of anticipated point mutations. This method has been applied in numerous studies in which tumors were examined for the presence of ras gene mutations. (1)