Figure 2.

Overview of single-molecule sequencers. The three most advanced single-molecule sequencing systems all carry out sequencing-by-synthesis using laser excitation to generate a fluorescent signal from labeled nucleotides, which is then detected using a camera. (a) In the Helicos BioSciences system [18], single nucleotides, each with a fluorescent dye attached to the base, are sequentially added. (b,c) In the Pacific Biosciences [35] and Life Technologies [41] systems, four different nucleotides, each with a different color dye attached to the phosphates, are continuously added. Background fluorescence is minimized differently in the three systems. (a) Helicos uses total internal reflectance fluorescence (TIRF) to create a narrow evanescent field of light in which the intensity of the light decays exponentially away from the glass surface. Only dyes that are in the TIRF evanescent field can fluoresce. (b) Pacific Biosciences uses a zero mode waveguide (ZMW), which limits illumination to a narrow region near the bottom of the well containing the polymerase. Only dyes near the opening of the ZMW can fluoresce. (c) Life Technologies uses fluorescence resonance energy transfer (FRET) between the initially absorbing quantum dot on the polymerase and the emitting dye on the nucleotide. Only dyes close to the polymerase-attached quantum dot can be excited by FRET and then fluoresce. For the three systems, DNA is immobilized for viewing over time by a surface-attached sequencing primer (Helicos (a)), by interaction with a surface-bound polymerase (Pacific Biosciences (b)), or by ligating to a surface-attached oligonucleotide (Life Technologies (c)). For Helicos (a), the polymerase is replaced after every cycle of nucleotide addition. For Life Technologies (c), the polymerase can be replaced on a given DNA molecule after each read is completed. For Pacific Biosciences (b), the polymerase cannot be replaced.