title: Fluorogenic polymerase, endonuclease, and ligase assays based on DNA substrates labeled with a single fluorophore.
abstract: This paper describes the development of homogeneous, fluorogenic polymerase, restriction endonuclease, and ligase assays based on the use of DNA substrate molecules labeled with a single fluorophore. All three enzymatic assays are based on the same observed phenomenon whereby the fluorescence intensity of hairpin-type oligonucleotides with a 5'single-stranded extension, labeled with a single fluorophore, changes when the distance of the dye from the 3' end of the molecule is altered as a result of the enzymatic transformation (i.e., polymerase extension, endonuclease hydrolysis, or ligation). The magnitudes of the observed fluorescence intensity changes range from 1.2-fold to 3.9-fold, and they are dependent on the type of dye used, its position within the substrate and product molecules, and the base composition surrounding the labeling site.
authors.affiliation: Life Technologies Corp., Carlsbad, CA 92008, USA. theo.nikiforov@lifetech.com
authors.forename: Theo T
authors.initials: TT
authors.lastname: Nikiforov
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D011088:DNA Ligases