Gene mapping and gene enrichment by the avidin-biotin interaction: use of cytochrome-c as a polyamine bridge.
A modification of previously described methods of electron microscopic gene mapping and of gene enrichment based on the avidin-biotin interaction is presented. The modification consists of coupling cytochrome-c instead of pentane diamine to the oxidized 2', 3' terminus of an RNA by Schiff base formation and BH-4 reduction. The RNA-cytochrome-c conjugate is purified by a simple chromatographic procedure; several biotins are attached to the cytochrome moiety by acylation. The extended arm between biotin and RNA gives efficient electron microscopic gene mapping of DNA:RNA-biotin hybrids with avidin-ferritin and avidin-polymethacylate sphere labels and efficient gene enrichment by buoyant banding of DNA:RNA-biotin:avidin-spheres in CsCl. A 1400 fold enrichment (thus, 25% pure) and a 90% yield of long Drosophila DNA strands with 5S RNA genes is achieved.
D001360:Avidin; D001710:Biotin; D055598:Chemical Phenomena; D002621:Chemistry; D002874:Chromosome Mapping; D003574:Cytochrome c Group; D004247:DNA / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008854:Microscopy, Electron / Q000379:methods; D012335:RNA, Ribosomal / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*
D003574:Cytochrome c Group; D012335:RNA, Ribosomal; D001360:Avidin; D001710:Biotin; D004247:DNA; D012343:RNA, Transfer