Enrichment of mutated K-RAS from human stool DNA by a combined magnetic capture hybridization and PCR-RFLP assay.

Research output: Contributions to collected editions/worksPublished abstract in conference proceedingsResearchpeer-review

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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
Translated title of the contributionAnreicherung von mutiertem K-RAS in humanen Stuhlproben mittels magnetic capture hybridization und PCR-RFLP (Restriktionsfragment-Längenpolymorphismus).
Original languageEnglish
Title of host publicationWissen teilen - Chancen nutzen : 28. Deutscher Krebskongress, Berlin, Februar 2008 und 3. Krebsaktionstag, Berlin, Februar 2008: Abstracts
EditorsM. Kaufmann, A. Rody, M. Bamberg
Number of pages1
Volume31
PublisherKarger Verlag für Medizin und Naturwissenschaften
Publication date01.01.2008
Pages59
ISBN (print)978-3-8055-8536-1
ISBN (electronic)978-3-8055-8537-8
DOIs
Publication statusPublished - 01.01.2008
Externally publishedYes
Event28. Deutscher Krebskongress - 2008: Wissen teilen - Chancen nutzen - Berlin, Germany
Duration: 20.02.200823.02.2008
Conference number: 28

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