(Step 1) A nucleotide forms an association with the template in the polymerase active site, (Step 2) causing an elevation of the fluorescence output on the corresponding color channel. (3) The fluorescent tag is liberated and diffuses out of the ZMW, thus ending the fluorescence pulse. (4) The polymerase translocates to the next position, and (5) the next cognate nucleotide binds the active site beginning the subsequent pulse.
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