Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension

Abstract

Objectives: Mutations of all three PAS genes, N-, H-, and KRAS, are identified mainly in codons 12, 13, and 61 of exons 2 and 3 in human cancers. Design and methods: DNA samples were isolated from 58 oral cancer and 106 colorectal cancer patients. Multiplex amplification of codons 12 and 13 of exon 2 and codon 61 of exon 3 of three PAS genes using two pairs of universal primers for exons 2 and 3 was performed in a single tube. The products were cleaned and split in three tubes. Each was subjected for primer extension using seven different-sized PAS primers for different PAS gene separately to detect base changes in codons 12, 13, and 61 of each RAS gene. Results: We compared the results with that from direct sequencing for detecting N-, H-, and KRAS mutations in 58 oral cancers and 106 colorectal cancers. The two methods yield identical results, but our method is superior to direct sequencing in terms the amount of work and time required. Conclusions: We presented a rapid method to detect codons 12, 13, and 61 mutations of N-, H, and KRAS genes in human cancers. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All fights reserved.