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Pyrosequencing as an Alternative to Single-Strand Conformation Polymorphism Analysis for Detection of N-ras Mutations in Human Melanoma Metastases

¹ AlbaNova University Center, Department of Molecular Biotechnology, Royal Institute of Technology (KTH), S-106 91 Stockholm, Sweden.

² Cancer Center Karolinska, Department of Oncology-Pathology, Radiumhemmet, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.

^aAuthor for correspondence. Fax 46-8-5537-8481; e-mail joakim.lundeberg{at}biotech.kth.se.

Background: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras.

Methods: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively.

Results: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified.

Conclusion: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.

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