Use of DNA purified in situ from cells embedded in agarose plugs for the molecular analysis of tk−/− mutants recovered in the L5178Y tk+/− 3.7.2C mutagen assay system

Abstract

We have reported that tk −/− mutants recovered in the mouse L5178Y tk +/− 3.7.2C mutagen assay have often lost the tk + allele. Allele loss in the tk −/− mutants is documented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk +/+ and tk +/− cell DNAs. For the routine screening of large- and small-colony tk −/− mutants DNAs for the absence of this genomic fragment, we have found that cells can be lysed in agarose plugs, and DNA of cells embedded in plugs can be purified, restricted with Nco I, electrophoresed, and analyzed on Southern blots without significant band distortion or diffusional loss of tk- specific fragments in the 2–7-kb range. Purification and restriction analysis of DNA in agarose plugs, originally developed to allow pulsed-field gel electrophoresis of very large DNA fragments, represents a convenient alternative to conventional DNA purification methods, allowing quantitative recovery of DNA from small numbers of cells, eliminating centrifugation, phenol extraction, and ethanol precipitation steps, and requiring smaller quantities of reagents.