Journal: PLOS One
Article Title: A denaturation-free protocol for in situ visualization of short nuclear DNA sequences using padlock probes with rolling-circle amplification
doi: 10.1371/journal.pone.0335619
Figure Lengend Snippet: (A) Workflow of dfPP. (B, C and D) Detection of DYZ1 (3,000 copies in chrY, red signals) in male PBMC (B), male Jurkat cells (C) and female GM12878 cells (D) without or with prior standard denaturation (Den – or Den + , respectively). The areas demarcated by white squares in the top images are shown at greater magnification below. White arrow heads indicate RCA signals located outside nuclei. (E) Detection of DYZ1 in Jurkat cells with combined padlock probe hybridization and ligation at the indicated temperatures, followed by RCA. White arrow heads indicate smaller RCA signals than the main signals. (F) Detection of DYZ1 in Jurkat cells by padlock probe hybridization for 30 min, followed by washing and then ligation for 30 min (left, 30/30 min) or by simultaneous hybridization and ligation for either 30 or 60 min (middle and right, 30 min and 60 min). (G) Detection of ssDNA (ss) and dsDNA (ds) forms of the M13 phage genome immobilized on slides. A nick was induced in the supercoiled circular dsDNA M13 by the nickase Nb.BtsI (Nicked). Samples omitting any types of M13 were treated as negative controls (M13 -). The white circles in enlarged images indicate fluorescent dots. The dot plot indicates numbers of fluorescent dots (RCA products) per a microscopy field (n = 6). Asterisk indicates p < 0.05. Scale bars in (B) and (G) and in other magnified images represent 100 μm and 20 μm respectively. Nuclear DNA is stained in blue except for in (G).
Article Snippet: One nM ssDNA M13 (N4040S, New England Biolabs), dsDNA supercoiled or nicked M13 DNA, serving as ligation targets, were incubated with 10 nM padlock probes, 0.8 U/μl Taq ligase and a biotin-conjugated RCA primer 2 (30 nM) at 55°C for 2 h. The reactions were diluted 200-fold with buffer C (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA (15575020, Thermo Fisher), 1 M NaCl and 0.05% Tween20) and added to streptavidin-coated slides (custom-made by TRIDIA) with FlexWell incubation chambers (204916, Grace Bio-Labs) for 1 h at room temperature.
Techniques: Hybridization, Ligation, Microscopy, Staining
New England Biolabs is a verified supplier 
New England Biolabs manufactures this product 
