title: Nicking enzyme-based internal labeling of DNA at multiple loci.
abstract: The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by Förster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3-5 d), whereas the labeling itself takes 4-6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.
authors.affiliation: Biotechnology Center, Dresden University of Technology, Dresden, Germany.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Luzzietti
authors.forename: Sabine
authors.initials: S
authors.lastname: Knappe
authors.forename: Isabel
authors.initials: I
authors.lastname: Richter
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D053904:DNA Breaks, Single-Stranded; D031541:Fluorescence Resonance Energy Transfer; D009841:Oligonucleotides / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods
chemical_list: D009841:Oligonucleotides; D004247:DNA