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

Creators: Pellegrini, Maria; Holmes, David S.; Manning, Jerry

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.

Additional Information

© 1977 Oxford University Press. Received July 5, 1977. This work was supported by grant GM 22207 from the National Institutes of Health and the Damon Runyon Memorial Fund (D.S.H.). M. Pellegrini is a Lievre fellow of the California division, American Cancer Society. We wish to thank L. Zimmerman and D. Fouts for helpful discussions; M. Grinder for preparation of nucleic acids; S. Krassner and W. Leon for preparation of L. donovani cells and rRNA. We particularly wish to thank Dr. Norman Davidson, in whose laboratory some of this work was performed, for his generosity and helpful council.

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