TY - CHAP

T1 - Enrichment of mutated K-RAS from human stool DNA by a combined magnetic capture hybridization and PCR-RFLP assay.

AU - Schneider, Mandy

AU - Steinberg, Pablo

AU - Scholtka, Bettina

N1 - Conference code: 28

PY - 2008/1/1

Y1 - 2008/1/1

N2 - Introduction: Although many techniques for DNA mutation analysis areavailable, most of them lack the sensitivity to detect alterations in human stoolDNA at a comparable level to that present in tissues. In faeces usually onemutated 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 toenrich the mutated K-RAS from a mixture of wild-type and mutant DNA instool and to compare the achieved detection rate with that of an establishedPCR-RFLP method.Methods: A combined magnetic capture hybridization and PCR-RFLP assayto detect K-RAS codon 12 or 13 mutations was developed. Briefly, two PCRreactions with an intermediate restriction digest followed by a hybridizationreaction 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 patientswith colon or rectum carcinomas or prestages were analyzed. All observedmutations were confirmed by sequencing.Results: The combination of restriction digest and magnetic capturehybridization led to a 100-fold increased detection limit for K-RAS mutationsin both, codons 12 and 13, when compared to the conventional PCR-RFLPassay. With the conventional PCR-RFLP assay it was not possible to detectalterations in stool samples of patients with known K-RAS gene mutations intheir corresponding preneoplastic or neoplastic intestinal tissues. In contrast,by using the combined magnetic capture hybridization and PCR-RFLP assayK-RAS mutations could be found in stool samples as previously observed inthe corresponding biopsies.Conclusion: The combined magnetic capture hybridization and PCR-RFLPassay leads to an enrichment of mutated K-RAS and is sensitive enough todetect K-RAS codon 12 or 13 mutations in stool DNA from patients with pre-neoplastic and neoplastic intestinal lesions

AB - Introduction: Although many techniques for DNA mutation analysis areavailable, most of them lack the sensitivity to detect alterations in human stoolDNA at a comparable level to that present in tissues. In faeces usually onemutated 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 toenrich the mutated K-RAS from a mixture of wild-type and mutant DNA instool and to compare the achieved detection rate with that of an establishedPCR-RFLP method.Methods: A combined magnetic capture hybridization and PCR-RFLP assayto detect K-RAS codon 12 or 13 mutations was developed. Briefly, two PCRreactions with an intermediate restriction digest followed by a hybridizationreaction 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 patientswith colon or rectum carcinomas or prestages were analyzed. All observedmutations were confirmed by sequencing.Results: The combination of restriction digest and magnetic capturehybridization led to a 100-fold increased detection limit for K-RAS mutationsin both, codons 12 and 13, when compared to the conventional PCR-RFLPassay. With the conventional PCR-RFLP assay it was not possible to detectalterations in stool samples of patients with known K-RAS gene mutations intheir corresponding preneoplastic or neoplastic intestinal tissues. In contrast,by using the combined magnetic capture hybridization and PCR-RFLP assayK-RAS mutations could be found in stool samples as previously observed inthe corresponding biopsies.Conclusion: The combined magnetic capture hybridization and PCR-RFLPassay leads to an enrichment of mutated K-RAS and is sensitive enough todetect K-RAS codon 12 or 13 mutations in stool DNA from patients with pre-neoplastic and neoplastic intestinal lesions

KW - Biology

KW - PCR

KW - DNA

U2 - 10.1159/000115424

DO - 10.1159/000115424

M3 - Published abstract in conference proceedings

SN - 978-3-8055-8536-1

VL - 31

T3 - Onkologie

SP - 59

BT - Wissen teilen - Chancen nutzen

A2 - Kaufmann, M.

A2 - Rody, A.

A2 - Bamberg, M.

PB - Karger Verlag für Medizin und Naturwissenschaften

Y2 - 20 February 2008 through 23 February 2008

ER -