Preparation and isolation of covalently closed circular rDNA molecules from DNA of Xenopus laevis.
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.
D000818:Animals; D004247:DNA; D004270:DNA, Circular / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011118:Polynucleotide Ligases; D014981:Xenopus
D004270:DNA, Circular; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004259:DNA-Directed DNA Polymerase; D011118:Polynucleotide Ligases