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Fig. 1 | BMC Molecular Biology

Fig. 1

From: 3C-digital PCR for quantification of chromatin interactions

Fig. 1

3C-dPCR workflow. A TaqMan probe and primer design. The locations of the two possible interaction fragments (a and b) are shown (black rectangle). Restriction sites used in the 3C assay are depicted as small vertical bars in black. The relative positions of anchor primer (black arrow), the TaqMan probe (F-Z-Q) and test primers (grey arrows) are also depicted. F fluorophore, Z internal quencher, Q quencher. B Three essential steps of 3C assays: 1. Interacting chromatin segments are cross-linked by formaldehyde. 2. Cross-linked chromatins are digested by a selected restriction enzyme. 3. Cross-linked fragments undergo intra-molecular ligation. C Principle of 3C-dPCR. The reaction mixture containing 3C DNA is prepared and partitioned into thousands of reaction wells. Due to significant dilution, each reaction well receives 0–1 target ligation products. After PCR amplification, the fluorescence signals are imaged and copy numbers of target ligations are reported as copies/μL. In the 3C-dPCR reaction and partition steps, curved lines in blue, red, black, grey and purple curve in the circle represent the different DNA molecules, including ligation products in 3C libraries. In the amplification step, the blue dot (well) shows target amplification signal; the red dots (wells) indicate the genome copy number signal; the green dot (well) displays the overlap of target and genome copy number signals

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