Single molecule real time sequencing (also known as SMRT) is a parallelized single molecule DNA sequencing by synthesis technology developed by Pacific Biosciences. Single molecule real time sequencing utilizes the zero-mode waveguide (ZMW), developed in the laboratories of Harold G. Craighead and Watt W. Webb[1] at Cornell University. A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA (also known as a base) being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
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The DNA sequencing is done on a chip that contains many ZMWs. Inside each ZMW, a single active DNA polymerase with a single molecule of single stranded DNA template is immobilized to the bottom through which light can penetrate and create a visualization chamber that allows monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a phospho-linked nucleotide incorporated by the DNA polymerase is detected as the DNA synthesis proceeds which results in the DNA sequencing in real time.
For each of the nucleotide bases, there are four corresponding fluorescent dye molecules that enable the detector to identify the base being incorporated by the DNA polymerase as it performs the DNA synthesis. The fluorescent dye molecule is attached to the phosphate chain of the nucleotide. When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is cleaved off with the phosphate chain as a part of a natural DNA synthesis process during which a phosphodiester bond is created to elongate the DNA chain. The cleaved fluorescent dye molecule then diffuses out of the detection volume so that the fluorescent signal is no longer detected. [2]
The zero-mode waveguide (ZMW) is a nanophotonic confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate.[3] The ZMW holes are ~70 nm in diameter and ~100 nm in depth. Due to the behavior of light when it travels through a small aperture, the optical field decays exponentially inside the chamber.[4] The observation volume within an illuminated ZMW is ~20 zeptoliters (20 X 10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.
Pacific Biosciences expects to commercialize SMRT sequencing in 2010 or 2011. The prototype of the SMRT chip contains ~3000 ZMW holes that allow parallelized DNA sequencing. Each of the ZMW holes produces approximately 1,500 bp (base pair) read lengths at a speed of 10 bp per second.
Single molecule real time sequencing will be applicable for a broad range of genomics research, namely: