FLUOROMETRIC ASSAY FOR DNA-POLYMERASES AND REVERSE-TRANSCRIPTASE

We report a quick, easy and inexpensive fluorometric assay that measur es the activity of replication enzymes using Pico-Green(TM). The syste ms tested include replication of the natural template M13 Gori by E. c oli DNA polymerase III holoenzyme and the replication of a synthetic h omopolymer by human immunodeficiency virus reverse transcriptase. A di rect comparison of the fluorometric assay with the conventional isotop ic assay shows that the fluorometric assay accurately reflects the ext ent of replication. By performing the assay reactions directly in 96-w ell plates and using a fluorescence plate reader to determine the exte nt of reaction, the time required to measure replication activities is significantly shortened.