Mutational analysis of human NRAS genes in malignant melanoma: Rapid methods for oligonucleotide hybridization and manual and automated direct sequencing of products generated by the polymerase chain reaction

Abstract

Three methods to detect single base mutations in codon 61 of the human NRAS gene from human melanoma DNA are described and compared: oligonuceotide hybridization analysis and direct manual and automated sequence analysis. Point mutations are detected by oligonucleotide hybridization and direct manual and direct automated sequence analysis of in vitro amplified genomic DNA. Heterozygosity for mutant alleles is reliably detected by oligonucleotide hybridization and by direct manual, but not by direct automated, sequence analysis. Generating single‐stranded DNA via “asymmetric polymerase chain reaction (PCR)” and utilizing α³⁵S‐dATP as radiolabel for manual sequencing and fluorescent‐dye labeled primers for automated sequencing (Applied Biosystems, Inc.), we can obtain sequence information from either strand. The use of several of these methodologies to detect single base changes in the human NRAS gene is illustrated. In addition, the use of these and other related techniques to define the involvement of RAS oncogenes in human melanomas more precisely is reviewed.