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

Abstract

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 A 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 × 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.