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

doi:10.1093/nar/4.9.2961

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

Life Sciences School

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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 language	English
Pages (from-to)	2961-2974
Number of pages	14
Journal	Nucleic Acids Research
Volume	4
Issue number	9
DOIs	https://doi.org/10.1093/nar/4.9.2961
Publication status	Published - Sep 1977

ASJC Scopus subject areas

Genetics

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@article{f7565c2ac86649538951c67625d894cb,

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

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.",

author = "Maria Pellegrini and Holmes, {David S.} and Jerry Manning",

note = "Funding Information: ACKNOWLEDGEMENTS 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.",

year = "1977",

month = sep,

doi = "10.1093/nar/4.9.2961",

language = "English",

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journal = "Nucleic Acids Research",

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AU - Pellegrini, Maria

AU - Holmes, David S.

AU - Manning, Jerry

N1 - Funding Information: ACKNOWLEDGEMENTS 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.

PY - 1977/9

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N2 - 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.

AB - 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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JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

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ER -

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

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