Rapid and reliable detection of N-ras mutations in acute lymphoblastic leukemia by melting curve analysis using LightCycler technology

We applied a new strategy for the detection of N-ras gene mutations based o n LightCycler technology. We designed two sets of amplimers and internal hy bridization probes representing N-ras codons 12/13 and codon 61, respective ly. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) pati ents (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were ampli fied, followed by the analysis of the melting temperatures of the PCR produ cts on the LightCycler. PCR products exhibiting an abnormal melting charact eristic were directly sequenced. Sequence analyses unravelled nucleotide su bstitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-ras mutations (10%) is compatible with p revious reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.