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

ABI PRISM 310 automated sequencer, Applied Biosystems, Germany).


Results

The calculated melting profile of the generated PCR fragment shows that it contains three melting domains. The putative mutations were in the second melting domain, which could not be analyzed without a GC clamp (the most stable domain) (Figure 1). The difference in melting profiles of the mutant and wt K-ras PCR-products should allow separation via CDGE (Figure 2). As controls we generated mutated sequences by PCR and cloning. Figure 3 shows a 2050% denaturant perpendicular DGGE gel run to determine the optimal denaturant concentration for the CDGE analysis, which was determined to be 35% (Figure 3). A 35% CDGE gel was run with samples from all mutations of codon 12 leading to an amino acid exchange and wt PCR products (Figure 4).

We analyzed 1,069 samples of 71 pancreases. Mutations were detected in 16 samples of 12 pancreases. With the exception of 2 samples of normal duct cells (243 screened) all the mutation-positive samples were from lesions (605 screened). All acinar cells screened (221) were negative. Figure 5 shows a typical result with one of the positive samples. In contrast to this low frequency, we could detect K-ras point mutations in more than 95% of ductal adenocarcinoma of the pancreas (data not shown).


Discussion

Mutations of K-ras were thought to be quite rare in pancreases free of ductal adenocarcinoma. However, we detected mutations in 16 samples from normal pancreases. This finding raises the question which role K-ras mutations play in the series of genetic alterations leading to ductal adenocarcinoma
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