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, Jerry Manning

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2961-2974
Number of pages14
JournalNucleic Acids Research
Volume4
Issue number9
DOIs
Publication statusPublished - Sep 1977

ASJC Scopus subject areas

  • Genetics

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