SMRT Sequencing Method

If you enjoyed the story of Juan and Estevan, you are already on to being an expert on error distribution in next-generation sequences generated by PacBio Instruments. The animals avestruz, cerdo, ganso and toro represent As, Cs, Gs and Ts. Juan’s list kept at the pond (ACTGTGATGGCCAA…..) is the actual sequence, whereas Estevan’s list noted from the top of Redwood tree is the sequence, as it comes out of the instrument.

At the heart of PacBio’s Single Molecule Real Time (SMRT) technology lies a tiny structure known as a zero mode waveguide (ZMW). It is a cylindrical hole etched on a silicon chip. The size is so small that the hole can carry only one DNA molecule. A typical silicon chip consists of thousands of such holes, each carrying a different DNA molecule.

A DNA polymerase is affixed at the bottom of each ZMW. When a DNA molecule passes through the polymerase, DNA synthesis takes place one nucleotide at a time, not too differently from the animals passing through the pond. The DNA bases A, C, G and T floating around carry fluorescent dyes of different colors, and so the incorporation of each nucleotide into the chain results in emission of light pulse of the corresponding color. Those colored pulses are detected by an optical sensor linked to the ZMW.