Figure 2: Principle of single molecule-real time sequencing. A) A single molecule of DNA template-DNA polymerase complex is immobilized at the bottom of ZMW illuminated by laser light from the bottom. THE ZMW enables detection of the each incorporated phospholinked nucleotide by the polymerase against the bulk background of nucleotides. B) Schematic representation of the phospholinked dNTP incorporation cycle, with a corresponding expected time trace of detected fluorescence intensity from the ZMW. (1) Cognate association of a phospholinked nucleotide with the template in the polymerase active site, (2) Increased fluorescence output on the corresponding color channel. (3) Formation of a phosphodiester bond liberates the dye-linkerpyrophosphate product that diffuses out of the ZMW, and ends the fluorescence pulse. (4) Translocation of the polymerase to the next position, and (5) binding of the next cognate molecule to the active site, thereby beginning the subsequent pulse (Figure reproduced from Eid et al with permission [24]).