In Vitro Iodination of Low Complexity Nucleic Acids without Chain Scission

In vitro TICl₃ catalyzed iodination of DNA may be performed at 60 °C, pH 4.7, in concentrated NaClO₄ solutions where double-stranded DNA is unstable. Other denaturing solvents inhibit iodination. The use of a denaturing solvent prevents DNA renaturation during the iodination procedure. The iodination reaction takes no more than 5 min with 15-20% of input iodide found in iodocytosine. The second heating step, to remove labile iodine, is performed at pH 7 under renaturation conditions to minimize depurination and other damage. A quantitative determination of chain scission accompanying iodination has been made. The single-strand molecular weight of the product DNA is 5 × 10⁶/percent cytosine as iodocytosine. When the iodide to cytosine ratio in the reaction mixture is 0.02 or less, A DNA may be labeled without significant chain scission as assayed by alkaline sedimentation velocity. A Hind III digest of iodinated, renatured, and endonuclease S1 treated λ DNA has a band pattern identical with unlabeled DNA upon agarose gel electrophoresis. Doublestranded RNA may also be labeled in concentrated NaClO₄ solutions.