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Nucleic Acids Research, 1979, Vol. 7, No. 6 1567-1578
© 1979

Articles

Restriction endonuclease mapping by crossed contact hybridization: the ribosomal RNA genes of Achlya ambisexuahis

Charles E. Rozek and Willam E. Timberlake

Department of Biological Sciences, Wayne State University Detroit, MI 48202, USA

Received September 24, 1979. A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RHA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure facilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.


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