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(American Journal of Pathology. 2003;162:737-746.)
© 2003 American Society for Investigative Pathology

Technical Advance

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Karl Sotlar^*, Luis Escribano, Olfert Landt, Stefanie Möhrle^*, Sonia Herrero, Antonio Torrelo, Ulrich Lass, Hans-Peter Horny^¶ and Burkhard Bültmann^*

From the Institute of Pathology,^* University of Tübingen, Tübingen, Germany; the Servicio de Hematologia, Mast Cell Unit, Hospital Ramón y Cajal, Madrid, Spain; the Department of Dermatology, Hospital del Niño Jesús, Madrid, Spain; TIB Molbiol Incorporated, Berlin, Germany; and the Institute of Pathology,^¶ University of Lübeck, Lübeck, Germany

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

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