The restriction site mutation assay: a review of the methodology development and the current status of the technique

The restriction site mutation (RSM) assay has been employed in our laborato ry, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous re striction enzyme sites and is, therefore, readily applicable to any sequenc ed gene and/or organism. The RSM assay has been applied in our laboratory i n various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumula ted by the RSM methodology in our hands and charts the developmental proces ses which have steadily improved the technique such that it is now applicab le as a sensitive genotypic mutation detection system. This paper also incl udes PCR primer sequences and restriction enzymes employed in mutational an alyses performed in the various species studied. We detail a variety of pro blems associated with the assay and the steps taken to solve them. The spec ific hurdles which have been overcome include the lack of quantitative data , the question of the contribution of DNA adducts to the induced mutation p rofile and the presence of false positives. Finally, the methods which have been developed to increase the sensitivity of the assay are also detailed. This paper describes our recommended RSM methodology, as it is routinely e mployed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in pres enting the developmental data on the RSM assay is to provide other research ers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.