The 14-3-3σ gene shows enhanced methylation in melanoma metastases compared with primary melanomas. Methylation of the 14-3-3σ gene was analyzed by methylation-specific PCR. For this purpose genomic DNA extracted from laser-microdissected melanoma tissues was bisulfite-treated and subjected to PCR amplification of the 14-3-3σ gene with primers specific for either unmethylated (U) or methylated (M) DNA alleles. (a) The percentage of methylated fraction is given for a representative set of tumors in each group after densitometric quantification of the PCR products separated by agarose gel electrophoresis. (b) Summary of the percentages of methylated alleles of all samples tested (15 primary melanomas, 20 lymph node metastases and 19 cutaneous metastases). Data are given as means +/- SD, P values represent statistical significance after applying Student's t-test. MM, primary melanomas; Meta (ln), lymph node metastases; Meta (s), cutaneous metastases.