( and primers as described by Imai et al...)Detection of Mutant K-ras in a Kindred With Hereditary Pancreatic Cancer by DGGE

and primers as described by Imai et al.³ Amplifications were carried out using 300 ng of DNA template in a buffer containing 10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3, with 2 units of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 200 M dNTPs, and 15 pmol of each primer in a total volume of 50 liters. Reactions were denatured for 3 minutes at 95 C, which was followed by thirty seven cycles with the following profile: 95 C (20s), 55 C (60s), 72 C (40s). The 110 bp product was run on a 3% agarose gel and visualized using ethidium bromide staining.

Successful samples were run on a 10% acrylamide 080% perpendicular DGGE gel at 150 V for 2 hours at 56 C, then stained with ethidium bromide to find the optimal conditions for parallel DGGE (Figure 1). This was determined to be 30%60%. Samples were then run on a parallel DGGE gel at 56 C for 4 to 5 hours at 150 V, and stained with ethidium bromide. Positive samples were sequenced using dye terminator chemistry and run on an ABI Prism (Perkin Elmer).

Results
Exon 1, containing codons 12 and 13, of the K-ras gene was examined for the presence of k-ras mutations by DGGE. Constitutional and pancreatic tissues or pancreatic juice in eight of the family members were evaluated. All samples that were positive by DGGE (Figure 2) were confirmed by DNA sequencing. Three of these individuals had K-ras mutations in codon 13 present in pancreatic cancer or precancerous tissue and three had mutations in codon 12. There was no evidence of K-ras mutation in the metastatic pancreatic cancer tissue from individual III.19, even when DNA was subcloned, or from the ERCP fluid or dysplastic tis
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