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 laboratory, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous restriction enzyme sites and is, therefore, readily applicable to any sequenced gene and/or organism. The RSM assay has been applied in our laboratory in various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumulated by the RSM methodology in our hands and charts the developmental processes which have steadily improved the technique such that it is now applicable as a sensitive genotypic mutation detection system. This paper also includes PCR primer sequences and restriction enzymes employed in mutational analyses performed in the various species studied. We detail a variety of problems associated with the assay and the steps taken to solve them. The specific hurdles which have been overcome include the lack of quantitative data, the question of the contribution of DNA adducts to the induced mutation profile 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 employed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in presenting the developmental data on the RSM assay is to provide other researchers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.
Jenkins
G J
GJ
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK. g.j.jenkins@swansea.ac.uk
Suzen
H S
HS
Sueiro
R A
RA
Parry
J M
JM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed
D004247:DNA; D004262:DNA Restriction Enzymes