Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method

Abstract

The principle of a DNA cloning procedure that directionally generates genomic DNA fragments 50-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high molecular weight genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 micrograms/ml), approximately equal to 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene has been tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated lambda phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.