Journal: PLoS ONE
Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ
Figure Lengend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.
Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.
Techniques: Labeling, Mass Spectrometry, Incubation, Activity Assay