Detection of amplified sequences in mammalian DNA by in-gel renaturation and SINE hybridization.
The presence of amplified sequences in mammalian DNA can be determined by in-gel renaturation of labeled restriction digests of total genomic DNA, provided that such sequences comprise at least 20-30 copies per haploid human or hamster genome or 40-50 copies per mouse genome. To detect amplified DNA in mouse cells at a lower level of amplification, a new procedure has been developed. This procedure combines in-gel DNA renaturation with Southern hybridization using a cloned probe containing a short interspersed repeated element (SINE). Using mouse cell lines containing amplified dihydrofolate reductase (DHFR) or c-Ki-ras genes as a model system, and the cloned B2 repeated sequence as a SINE probe, we have shown that this technique can detect gene amplification at a level as low as 10-15 copies of amplified DNA per haploid mouse genome. In-gel renaturation-SINE hybridization was used to assay for DNA amplification in different tissues and in different mouse strains. No tissue-specific DNA amplification has been detected, but comparison of B2-containing repeated fragments between three inbred mouse lines revealed strain-specific polymorphism.
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004351:Drug Resistance; D004586:Electrophoresis / Q000379:methods; D005782:Gels; D005784:Gene Amplification; D005796:Genes / Q000187:drug effects; D006238:Haploidy; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009693:Nucleic Acid Hybridization; D009695:Nucleic Acid Renaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D005782:Gels; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes; D008727:Methotrexate