Preparation of pure oligonucleotide-alkaline phosphatase conjugates.

The utility and sensitivity of enzyme-labeled oligonucleotide probes have been described (1, 2). However, most methods used for their preparation require the use of extensive column chromatography, polyacrylamide gel electrophoresis, or high-pressure liquid chromatography to yield a pure product (3-5). Commercial kits offer a simple alternative but typically 1) are expensive, 2) utilize freeze dried enzyme, 3) rely on size exclusion chromatography, which may yield an impure product. Here we describe methods for the conjugation of 5'-amine and 5'-thiol-labeled oligonucleotides to alkaline phosphatase. These methods employ inexpensive consumables to produce pure conjugates. Oligonucleotides were initially 5'-labeled during automated synthesis using