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Nucleic Acids Research, 1977, Vol. 4, No. 9 2961-2974
© 1977

Articles

Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences

Maria Pellegrini*, David S. Holmes{dagger} and Jerry Manning*

*Department of Molecular Biology, University of California Irvine, CA 92717, USA {dagger}Division of Chemistry and Chemical Engineering, California Institute of Technology Pasadena, CA 91125, USA

Received July 5, 1977. 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.


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