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Detection of K-ras Point Mutations in the Pancreas by Constant Denaturing Gel Electrophoresis Using the DCode System

Martin A.O.H. Menke*, Axel Reinecke-Lthge*, Barbara Mllmann*, Angela Kellner, Jutta Lttges*, Gnter Klppel*
* Department of Pathology and Department of Hematopathology, Christian- Albrechts University Kiel, Michaelisstrae 11, D-24105 Kiel, Germany.


Introduction

Genetic alterations/variations are the cause underlying many non-neoplastic and probably all neoplastic diseases. Today the number of reports associating genetic alterations with cancer1 and other diseases is rapidly increasing. In many cases the exchange of only one or a few bases, so called point mutations, give rise to severe disease.

We are specifically interested in ductal adenocarcinoma of the exocrine pancreas, which is the second most frequent cause of death by digestive tract cancer.2 Nearly all of these tumors bear a point mutation in codon 12 of the K-ras protooncogene.3, 4 We wanted to determine the frequency of these mutations in ductal lesions and associated tissues of tumor free pancreases. We used microdissection followed by a sensitive single step PCR regime to amplify the exon 1 of the K-ras gene. Point mutations were detected by constant denaturing gel electrophoresis (CDGE)5 using the DCode universal mutation detection system.


Materials and Methods

Archived, formalin-fixed, paraffin-embedded pancreas tissue samples of patients without pancreatic ductal adenocarcinoma were drawn from the files of the Institute of Pathology. Evaluation of conservation was done by histological routine procedures. A section was stained with Hematoxylin & Eosin and used as reference. Cells were microdissected from an immediate
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