Detection of amplified sequences in mammalian DNA by in-gel renaturation and SINE hybridization

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Abstract
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-rasgenes 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.