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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 C̱CT GGT…) for an enzyme ( Bst NI) (5′CC!WGG 3′) to differentiate between wild-type sequence (…GGA G̱CT GGT…) (during amplification, the G̱ is replaced by a C̱) 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.

Restriction Digest PCR (RD-PCR) for the Analysis of Gene Mutations. Application to Ki- ras