Biopolis Dresden Imaging Platform

Nicking enzyme-based internal labeling of DNA at multiple loci.

Luzzietti N, Knappe S, Richter I, Seidel R

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.

Fig.3 taken from Luzzietti et al, 2012.
  • Nat Protoc 2012 Apr 08;7(4):643-53
  • 2012
  • Cell Biology
  • 22402634
  • PubMed

Enabled by:

Back to list