Single-Molecule Real Time Sequencing (SMRT) uses something called zero-mode waveguide (ZMW) nanostructures, which are essentially small well-like structures on an aluminum layer. There is a whole array of these well nanostructures on the metal, and we will discuss what happens in one of these nanostructures. This technology is advantageous because it can read longer segments of DNA  up to around 1,000 bases long.

Diagram A shows the interior of a ZMW nanostructure, in which a DNA polymerase is attached. The detector of the optical imaging system is located under the nanostructure. Diagram B shows the incorporation of a DN5P and detection of the color of the fluorescent group. The fluorescent group is cut off only a few milliseconds after the base is attached, enough time for detection of the color by optical sensors.

  1. DNA polymerase attaches to the bottom of a ZMW nanostructure, and sequencing begins. This is possible when DNA sequenced, the polymerase reels in the DNA like a fishing line, instead of physically moving.
  2. Fixed in place, DNA polymerase adds modified nucleotides called deoxyribonucleoside pentaphosphate (dN5Ps) which, like those in first and second generation methods, have a fluorescent group at its end for each different base.
  3. An optical imaging system beneath the ZMW nanostructure can read the color of the fluorescent group after it has been attached. However, unlike second generation methods, the detection of this group happens WHILE the DNA polymerase is adding bases, and the fluorescent group is automatically cut off by the DNA polymerase as part of the natural reaction.
  4. DNA polymerase continues adding bases immediately after detection, without the need to pause. (34) (44)

Notice that the polymerase "pulls" on the DNA to sequence it.

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