@inbook{60689a172e4d412ab1ad2cb63ed4bf2f,
title = "Enrichment of mutated K-RAS from human stool DNA by a combined magnetic capture hybridization and PCR-RFLP assay.",
abstract = "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",
keywords = "Biology, PCR, DNA",
author = "Mandy Schneider and Pablo Steinberg and Bettina Scholtka",
note = "Ersch. als Zeitschriftensonderheft: Onkologie 2008;31(suppl 1):1–211 Abstracts; 28. Deutscher Krebskongress - 2008 : Wissen teilen - Chancen nutzen ; Conference date: 20-02-2008 Through 23-02-2008",
year = "2008",
month = jan,
day = "1",
doi = "10.1159/000115424",
language = "English",
isbn = "978-3-8055-8536-1 ",
volume = "31",
series = "Onkologie",
publisher = "Karger Verlag f{\"u}r Medizin und Naturwissenschaften ",
number = "Suppl. 1",
pages = "59",
editor = "M. Kaufmann and A. Rody and M. Bamberg",
booktitle = "Wissen teilen - Chancen nutzen",
address = "Switzerland",
}