Fragment analysis (deletion/insertion of single nucleotides)

A defect mismatch repair system is seen in approximately 15% of all colorectal carcinomas and they show a genome wide instability in repetitive sequences. Genes with repeats within their coding region are prone to mutation in these tumours.

Frame-shift mutations are detected by multiplex PCR and capillary electrophoresis on an Applied Biosystems^TM3730 DNA Analyzer. Mutations are verified by sequencing (Applied Biosystems^TM3730 DNA Analyzer). Mutation assays are established for 40 such target genes.

We recently identified frame-shift mutations in a novel target gene, WISP-3, in colorectal carcinomas with microsatellite instability (Thorstensen et al., 2001, Gastroenterology). Tumors C1141 and C1314 reveal heterozygous mutations in WISP-3. The box to the left illustrates analyses of the PCR products on an ABI PRISMTM310 Genetic Analyzer (the number in the blue boxes indicate the size of the PCR products), whereas the sequenced PCR products are shown to the right. Wt; wild type, mut; mutation. — We recently identified frame-shift mutations in a novel target gene, WISP-3, in colorectal carcinomas with microsatellite instability (Thorstensen *et al.*, 2001, Gastroenterology). Tumors C1141 and C1314 reveal heterozygous mutations in WISP-3. The box to the left illustrates analyses of the PCR products on an ABI PRISMTM310 Genetic Analyzer (the number in the blue boxes indicate the size of the PCR products), whereas the sequenced PCR products are shown to the right. Wt; wild type, mut; mutation.

In addition, fragment analysis is used to perform methylation specific PCR with fluorescently labeled primers.

Own publications using this methodology:

Thorstensen L, Holm R, Lothe RA, Tropé C, Carvalho B, Sobrinho-Simoes M, and Seruca R (2003). WNT-inducible signaling pathway protein 3, WISP-3, is mutated in microsatellite unstable gastrointestinal carcinomas but not in endometrial carcinomas. Gastroenterology, 124(1): 270-271.

Thorstensen L, Diep CB, Meling GI, Aagesen TH, Ahrens CH, Rognum TO, and Lothe RA (2001). WNT1 inducible signaling pathway protein 3, WISP-3, a novel target gene in colorectal carcinomas with microsatellite instability. Gastroenterology, 121(6): 1275-1280.