Document 0402
 DOCN  M9590402
 TI    Fluorometric measurement of reverse transcriptase activity with
       4',6-diamidino-2-phenylindole.
 DT    9509
 AU    Chavan SJ; Prochaska HJ; Molecular Pharmacology and Therapeutics
       Program, Memorial; Sloan-Kettering Cancer Center, New York, New York
       1002, USA.
 SO    Anal Biochem. 1995 Feb 10;225(1):54-9. Unique Identifier : AIDSLINE
       MED/95297704
 AB    We describe a rapid fluorometric assay for reverse transcriptase (RT)
       activity. After RT is incubated in the presence of poly(A).oligo(dT) and
       dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are
       added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI
       fluorescence, which is increased upon binding the RNA.DNA heteroduplex,
       is measured after 30 min and is linearly dependent on the enzymatic
       reaction time and the amount of active RT added to the enzyme assay. The
       increased fluorescence correlates well with the incorporation of
       [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the
       Klenow fragment using poly(dA).oligo(dT) did not result in increased
       fluorescence under conditions wherein incorporation of [alpha-32P]dTTP
       into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA]
       heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in
       a manner that allows for a perturbation of DAPI fluorescence. The
       relative specific activities of RT in crude preparations measured with
       the fluorometric assay were comparable to conventional isotopic enzyme
       assays as were determinations for the type of inhibition and the kinetic
       constants of purified RT with inhibitors such as zidovudine
       5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250
       WORDS)
 DE    Antiviral Agents/*PHARMACOLOGY  Comparative Study  Fluorescent Dyes
       HIV-1/ENZYMOLOGY  Indicators and Reagents  *Indoles  Kinetics
       Phosphorus Radioisotopes  Pyrazines/PHARMACOLOGY  Pyridines/PHARMACOLOGY
       Radioisotope Dilution Technique  Reverse Transcriptase/ANTAGONISTS &
       INHIB/*ANALYSIS/METABOLISM  Spectrometry, Fluorescence/METHODS  Support,
       Non-U.S. Gov't  Support, U.S. Gov't, P.H.S.  Thymine
       Nucleotides/PHARMACOLOGY  Zidovudine/ANALOGS & DERIVATIVES/PHARMACOLOGY
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).