Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences.
A method of enriching for long double-stranded segments of eukaryotic DNA carrying particular genes is described. A purified RNA coded for by the gene is covalently attached to biotin via the protein, cytochrome c. This modified RNA is hybridized to total nuclear, double-stranded DNA under conditions that allow the formation of R-loops. Avidin, which has a high affinity for biotin, is covalently attached to polymer spheres. The complexes of avidin-spheres with DNA:RNA-biotin R-loop hybrids band in CsCl at a much lower bouyant density than does free DNA. This density is a function of the length of DNA coupled per avidin-sphere. This method was used to prepare very long double-strands of DNA highly enriched in the coding sequences for the large rRNAs of D. melanogaster and L. donovani and the histone mRNAs of S. purpuratus.
D000818:Animals; D001360:Avidin; D001710:Biotin; D002467:Cell Nucleus / Q000032:analysis; D003574:Cytochrome c Group; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster; D005796:Genes; D005815:Genetic Code; D006657:Histones / Q000096:biosynthesis; D007891:Leishmania; D008722:Methods; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D012617:Sea Urchins
D003574:Cytochrome c Group; D006657:Histones; D012333:RNA, Messenger; D001360:Avidin; D001710:Biotin; D004247:DNA