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Single Molecule Real Time Sequencing (SMRT) is a process developed to mimic DNA replication in vivo. In a zero-mode waveguide, a single molecule of single stranded DNA and DNA polymerase are suspended. Fluorescently labeled nucleotides are then added to the waveguide and DNA polymerase begins replicating the single strand of DNA. When the polymerase adds a nucleotide from the solution, its fluorophore temporarily fluoresces and the signal is picked up by the waveguide. Each nucleotide is labeled with a different color, so as each nucleotide is added to the chain being created by the polymerase, the specific nucleotide sequence can be determined by the sequence of colors picked up by the detector.

A unique feature of SMRT sequencing is that once the polymerase attaches a nucleotide and the fluorophore emits a signal, the fluorophore detaches from the nucleotide and the DNA formed is completely viable and biocompatible. This indicates the possibility of de novo sequencing, or, the possibility of sequencing in the DNA’s natural environment. This technique also employs the natural speed with which DNA polymerase works within the body, thus providing the possibility that an entire human genome could be sequence with SMRT sequencing in under an hour.