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Main Title 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.
Author Applegate, M. ; Juhn, G. ; Moore, M. ; Hozier, J. ;
CORP Author Health Effects Research Lab., Research Triangle Park, NC. ;Florida State Univ., Tallahassee. Dept. of Biological Science.
Publisher c1990
Year Published 1990
Report Number EPA/600/J-90/440;
Stock Number PB91-177212
Additional Subjects Mutagens ; Thymidine kinase ; Deoxyribonucleic acids ; Mutagenicity tests ; Southern blotting ; Agar gel electrophoresis ; Alleles ; Mutation ; Cell line ; Reprints ;
Library Call Number Additional Info Location Last
NTIS  PB91-177212 Some EPA libraries have a fiche copy filed under the call number shown. 07/26/2022
Collation 7p
It has been reported that tk-/- mutants recovered in the mouse L5178Y TK+/- 3.7.2C mutagen assay have often lost the tk+ allele. Allele loss in 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-/- mutant DNAs for the absence of the genomic fragment, it has been 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- to 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. (Copyright (c) Elsevier Science Publishers B.V. (Biomedical Division).)