title: One-step assay for the quantification of T4 DNA ligase.
abstract: As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.
authors.affiliation: Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, (BBZ), Institute of Bioanalytical Chemistry, Leipzig University, Deutscher Platz 5, 04103, Leipzig, Germany.
authors.forename: Steffi
authors.initials: S
authors.lastname: Franke
authors.forename: Thomas
authors.initials: T
authors.lastname: Kreisig
authors.forename: Karin
authors.initials: K
authors.lastname: Buettner
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D000476:Alkanesulfonates / Q000737:chemistry; D001391:Azo Compounds / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D011088:DNA Ligases / Q000032:analysis / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D000476:Alkanesulfonates; D001391:Azo Compounds; C000598590:BHQ-1 compound; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D011088:DNA Ligases; D016650:Fluorescein-5-isothiocyanate