Introduction: Although many techniques for DNA mutation analysis are
available, most of them lack the sensitivity to detect alterations in human stool
DNA at a comparable level to that present in tissues. In faeces usually one
mutated DNA molecule among hundred to ten thousand wild-type DNA mol-
ecules does in fact exist. The aim of the project was to develop an assay to
enrich the mutated K-RAS from a mixture of wild-type and mutant DNA in
stool and to compare the achieved detection rate with that of an established
PCR-RFLP method.
Methods: A combined magnetic capture hybridization and PCR-RFLP assay
to detect K-RAS codon 12 or 13 mutations was developed. Briefly, two PCR
reactions with an intermediate restriction digest followed by a hybridization
reaction with a biotinylated single-strand DNA probe were performed.
Positive controls were derived from human carcinoma cell lines. Serial dilu-
tions of mutant and wild-type controls were prepared to determine the sensi-
tivity of the assay. Furthermore, a few stool samples and tissues from patients
with colon or rectum carcinomas or prestages were analyzed. All observed
mutations were confirmed by sequencing.
Results: The combination of restriction digest and magnetic capture
hybridization led to a 100-fold increased detection limit for K-RAS mutations
in both, codons 12 and 13, when compared to the conventional PCR-RFLP
assay. With the conventional PCR-RFLP assay it was not possible to detect
alterations in stool samples of patients with known K-RAS gene mutations in
their corresponding preneoplastic or neoplastic intestinal tissues. In contrast,
by using the combined magnetic capture hybridization and PCR-RFLP assay
K-RAS mutations could be found in stool samples as previously observed in
the corresponding biopsies.
Conclusion: The combined magnetic capture hybridization and PCR-RFLP
assay leads to an enrichment of mutated K-RAS and is sensitive enough to
detect K-RAS codon 12 or 13 mutations in stool DNA from patients with pre-
neoplastic and neoplastic intestinal lesions