Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.
Journal: 1976/February - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 1105573
Abstract:
A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.
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Proc Natl Acad Sci U S A 72(10): 3961-3965

Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.

Abstract

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.

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Selected References

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  • Wensink PC, Finnegan DJ, Donelson JE, Hogness DS. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. [PubMed] [Google Scholar]
  • Glover DM, White RL, Finnegan DJ, Hogness DS. Characterization of six cloned DNAs from Drosophila melanogaster, including one that contains the genes for rRNA. Cell. 1975 Jun;5(2):149–157. [PubMed] [Google Scholar]
  • Katz L, Kingsbury DT, Helinski DR. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591.[PMC free article] [PubMed] [Google Scholar]
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  • Randerath K. An evaluation of film detection methods for weak beta-emitters, particularly tritium. Anal Biochem. 1970 Mar;34:188–205. [PubMed] [Google Scholar]
Abstract
A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.
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