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

Maria Pellegrini | Zendy; David S. Holmes | Zendy; Jerry E. Manning | Zendy

Open Access

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

Author(s) -

Maria Pellegrini,

David S. Holmes,

Jerry E. Manning

Publication year - 1977

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/4.9.2961

Subject(s) - biology , dna , biotin , rna , microbiology and biotechnology , avidin , biochemistry , base pair , gene , nucleic acid thermodynamics

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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