Fluorometric assay for DNA polymerases and reverse transcriptase

Biotechniques. 1996 Oct;21(4):664, 666, 668, 670, 672. doi: 10.2144/96214st04.


We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using Pico-GreenTM. The systems tested include replication of the natural template M13 Gori by E. coli DNA polymerase III holoenzyme and the replication of a synthetic homopolymer by human immunodeficiency virus reverse transcriptase. A direct comparison of the fluorometric assay with the conventional isotopic assay shows that the fluorometric assay accurately reflects the extent of replication. By performing the assay reactions directly in 96-well plates and using a fluorescence plate reader to determine the extent of reaction, the time required to measure replication activities is significantly shortened.

MeSH terms

  • DNA-Directed DNA Polymerase / metabolism*
  • Fluorometry
  • Humans
  • RNA-Directed DNA Polymerase / metabolism*


  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase