We describe a method leading to the formation of closed circles of rDNA starting from total DNA of Xenopus laevis. Linear DNA molecules were digested with exonuclease 3 and self-annealed. Open circles were enriched and covalently closed by the simultaneous use of polynucleotide kinase, DNA polymerase and polynucleotide ligase. Closed circles of rDNA1 were shown to be alkali-resistant, to have higher density than linear molecules in cesium chloride density gradients containing ethydium bromide, and to have the sedimentation constant expected for a single repeat unit of rDNA comprehensive of its spacer.
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