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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 beta-globin alleles from an individual with beta-thalassaemia.

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

11/11/1985

Volume

13

Pages

7569 - 7578

Keywords

Alleles, Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, DNA Transposable Elements, DNA, Recombinant, Humans, Nucleic Acid Hybridization, Plasmids, Thalassemia