Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment.
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.
D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004274:DNA, Recombinant / Q000378:metabolism; D006801:Humans; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013789:Thalassemia / Q000235:genetics
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes