Open AccessFluorometric Assay for DNA Polymerases and Reverse Transcriptase
Author(s) -
Mark Seville,
Anthony B. West,
Millard G. Cull,
Charles S. McHenry
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96214st04
Subject(s) - reverse transcriptase , dna polymerase , replication (statistics) , polymerase , biology , microbiology and biotechnology , dna replication , dna , polymerase chain reaction , rna directed dna polymerase , nucleotidyltransferase , enzyme , virology , genetics , gene , biochemistry , rna
We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using Pico-Green TM . 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.