Detection in situ of foreign DNA in eukaryotic cells.
A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D010584:Bacteriophage phi X 174 / Q000235:genetics; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002999:Clone Cells / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D004274:DNA, Recombinant / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D007739:L Cells / Q000378:metabolism; D051379:Mice
D004274:DNA, Recombinant; D004279:DNA, Viral; D004247:DNA