Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences.
Nucleic Acids Res, 1977/9;4(9):2961-73.
Pellegrini M, Holmes DS, Manning J
PMID: 198749
Impact factor: 19.16
Abstract
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.
MeSH terms
Animals; Avidin; Biotin; Cell Nucleus; Cytochrome c Group; DNA; Drosophila melanogaster; Genes; Genetic Code; Histones; Leishmania; Methods; Microscopy, Electron; Nucleic Acid Conformation; Nucleic Acid Hybridization; RNA, Messenger; Sea Urchins
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