J. Mol. Biol.
(1975) 98, 503-517
Detection of Specific Sequences
DNA FragmentsSeparated by Gel Electrophoresis
"E,. 1~. SOUTHERN
Medical Research Council Mammalian Genome UnitDeIaartment of ZoologyUniversity of EdinburghWest Mains Road, Edinburgh, Scotland(Received 3 March 1975, and in revised form 26 June 1975)
This paper describes a method of transferring fragments of DNA from agarosegels to cellulose nitrate filters. The fragments can then be hybridized to radio-active RNA and hybrids detected by radioautography or fluorography. Themethod is illustrated by analyses of restriction fragments complementary toribosomal RNAs from
Esoherichia coli and Xenopus lacvis,
and from severalmammals.
Since Smith and his colleagues (Smith & Wilcox, 1970; Kelly & Smith, 1970) showedthat a restriction endonuclease from
makes double-strandedbreaks at specific sequences in DNA, this enzyme and others with similar propertieshave been used increasingly for studying the structure of DNA. Fragments producedby the enzymes can be separated with high resolution by electrophoresis in agaroseor polyacrylamide gels. For studies of sequences in the DNA that are transcribed intoRNA, it would clearly be helpful to have a method of detecting fragments in the gelthat are complementary to a given RNA. This can be done by slicing the gel, elutingthe DNA and hybridizing to RNA either in solution, or after binding the DNA tofilters. The method is time consuming and inevitably leads to some loss in the resolv-ing power of gel electrophoresis. This paper describes a method for transferringfragments of DNA from strips of agarose gel to strips of cellulose nitrate. After hybri-dization to radioactive RNA, the fragments in the DNA that contain transcribedsequences can be detected as sharp bands by radioautography or fluorography of thecellulose nitrate strip. The method has the advantages that it retains the high resolv-ing power of the gel, it is economical of RNA and cellulose nitrate filters, and severalelectrophoretograms can be hybridized in one day. The main disadvantage is thatfragments of 500 nucleotide pairs or less give low yields of hybrid and such fragmentswill be under-represented or even missing from the analysis.2. Materials,
EcoRI prepared according to the method of u (1971) was a gift of K.Murray. HaeIII prepared by a modification of the method of Roberts (unpublisheddata) was a gift of H. J. Cooke.503