Genotypic analysis of N-ethyl-N-nitrosourea-induced mutations by Taq I restriction fragment length polymorphism/polymerase chain reaction in the c-H-ras1 gene

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 12; pp. 5331 - 5335
• Main Authors: CHIOCCA, S. M, SANDY, M. S, CERUTTI, P. A
• Format: Journal Article
• Language: English
• Published: Washington, DC National Acad Sciences 15-06-1992; National Academy of Sciences of the United States of America; National Academy of Sciences
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; c-H-ras1 gene; c-Ha-ras-1; Cells, Cultured; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - isolation & purification; DNA-Directed DNA Polymerase; Ethylnitrosourea - pharmacology; Eukaryotes; Fibroblasts - physiology; Fundamental and applied biological sciences. Psychology; Genes, ras; Genetic technics; Genetics; Genotype; genotypes; Humans; induction; man; Methods. Procedures. Technologies; Molecular Sequence Data; Mutagenesis; Mutant screening; mutation; N-ethyl-N-nitrosourea; Oligodeoxyribonucleotides; oncogenes; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; restriction fragment length polymorphism; Skin; Taq Polymerase; Human; Polymerase chain reaction; Restriction fragment length polymorphism; Investigation method; Enzyme; Cleavage site; C-Onc gene; Endodeoxyribonuclease TaqI; Mutation; Fibroblast
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.12.5331

In genotypic mutation analysis DNA sequence changes are determined without the in vivo or in vitro selection of phenotypically altered cells. We have studied the induction of base-pair changes by N-ethyl-N-nitrosourea in Taq I endonuclease recognition site 2508-2511 (TCGA) of the c-H-ras1 gene in human fibroblasts by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method. This site contains the four bases, and all 12 possible single base-pair changes can be monitored. The transition of guanine to adenine at position 2510 was the major mutation detected by lambda plaque oligonucleotide hybridization and quantitative sequence analysis of the RFLP/PCR products. It involves the G residue of the CpG sequence of the coding strand. Data calibration with an internal mutant standard indicates that absolute frequencies for this transition lie in the range of 4-12 x 10(-7). The present study documents the capacity of the RFLP/PCR approach to measure mutagen-induced base-pair changes in a specific gene sequence without the selection of a phenotypically altered cell.