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. 2005 Jan 11;92(2):405–412. doi: 10.1038/sj.bjc.6602319

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Copyright © 2005 Cancer Research UK

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Detection of Ki-ras point mutations in tissue samples. DNA from paraffin-embedded tissue samples of four different patients with pancreatic cancer were extracted by spin column technology after overnight digestion with proteinase K. After rapid cycle amplification of the eluted DNA in the presence of the valine mutation-specific hybridisation probes and wild-type-specific PNA, the melting curves were analysed by the LightCycler software (version 3.5). Temperature transition rate was 0.3°C s⁻¹ in the range of 40–95°C. PCR experiments were performed twice. Melting temperatures (T_m): wild-type DNA: 69.5°C, valine DNA 72.1°C and unknown mutation 68.5°C. 1: Nontemplate control (NTC); 2: 1 μg wild-type DNA (WT) without PNA; 3–7: tissue samples of patients. From patient no. 4, two different samples (4A and 4B) were analysed; 16: 100 pg Val DNA/1 μg WT DNA in the presence of PNA.