Restriction digest PCR (RD-PCR) for the analysis of gene mutations. Application to Ki-ras

The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, enrichment PCR must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5'primer (...GGA CCT GGT...) for an enzyme (BstNI) (5'CC!WGG 3') to differentiate between wild-type sequence (...GGA GCT GGT...) (during amplification, the G is replaced by a C) and mutated sequences (―...GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the secound round altogether. The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.