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Nucleic Acids Research, 1985, Vol. 13, No. 21 7569-7578
© 1985

Articles

Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment

R.D. Nicholls, A.V.S. Hill, J.B. Clegg and D.R. Higgs

MRC Molecular Hacmatology Unit, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital Headington, Oxford OX3 9DU, UK

Received September 16, 1985. Revised October 15, 1985. Accepted October 15, 1985.

We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10–1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human {alpha}-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both ß-globin alleles from an individual with ß-thalassaemia.


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