Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions.
A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.
Bragin
A G
AG
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. abgicg@mail.ru
Glushkov
S A
SA
Ivanov
M K
MK
Krasnov
A A
AA
Dymshits
G M
GM
D001671:Biochemistry / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019914:Taq Polymerase; D013696:Temperature; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D005456:Fluorescent Dyes; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D005092:Exonucleases