The restriction site mutation RSM assay see Steingrimsdottir et al. H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, Ž. Ž w
T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of
genetic alteration in man, Mutat. Res. 353 1996 pp. 109–121 for a review has been developed as a genotypic mutation Ž . x .
detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will
report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited
sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data
was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus
allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased
through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons,
presumably due to differential selective pressure within genes G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced w
restriction site mutation RSM analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous Ž . p53 intron
sequences, Mutagenesis 12 1997 pp. 117–123 . This increased sensitivity was examined by applying the RSM assay to Ž . x
analyse the persistence of N-ethyl-N-nitrosourea ENU -induced mutations in mice testes. Germ line mutations were sought Ž .
in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the
intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations
as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation
persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide 4-NQO -induced DNA mutations Ž .
in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the
concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that
early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq
polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this
time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes
produced in the fibroblasts by the in vivo processing of DNA adducts
