High-efficiency preparation of DNA from limiting quantities of eukaryotic cells for hybridization analysis.
This report describes a DNA preparation method which allows the detection of single-copy genes in samples of as few as 6000 eukaryotic cells. The technique uses proteinase K digestion in detergent and low-gelation-temperature agarose followed by solidification of the agarose and removal of the detergent by diffusion. RNase and restriction enzyme digestion are carried out in solution after remelting the agarose. The procedure can be performed successfully with mammalian cells in suspension, with parasitic protozoa and with pieces of mammalian tissue weighing less than 1 mg. Numerous samples can be processed simultaneously using frozen as well as fresh material.
D000818:Animals; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D003902:Detergents; D004587:Electrophoresis, Agar Gel / Q000379:methods; D019286:Endopeptidase K; D010450:Endopeptidases; D005796:Genes; D051379:Mice; D009693:Nucleic Acid Hybridization; D012260:Ribonucleases / Q000378:metabolism
D003902:Detergents; D004247:DNA; D012260:Ribonucleases; D004262:DNA Restriction Enzymes; D010450:Endopeptidases; D019286:Endopeptidase K