- Single Molecule Real Time Sequencing
Single Molecule Real Time Sequencing (also known as SMRTTM) is a parallelized single molecule
DNA sequencingby synthesis developed by [http://www.pacificbiosciences.com/index.php Pacific BiosciencesTM] . Pacific Biosciences is a company based in Menlo Park, California, which was founded in 2004. The Single Molecule Real Time sequencing utilizes the Zero-mode waveguide (ZMW), developed in the laboratory of Dr. Harold G. Craighead [M.J. Levene, J. Korlach, S.W. Turner, M. Foquet, H.G. Craighead, W.W. Webb, Zero-Mode Waveguides for Single-Molecule Analysis at high concentrations. Science. 299 (2003) 682-686] at Cornell University. A DNA polymeraseis affixed at the bottom of a ZMW with a single molecule of single stranded 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 being incorporated by DNA polymerase. Each of the four bases of DNAmolecules is attached to one of the four different fluorescent dyes at the phosphate chain of DNA. When a nucleotide is incorporated by the DNA polymerase, the fluorescent-tagged phosphate chain is cleaved off and is diffused out of the area in the ZMW where is not illuminated, thus, not observable. A detector detects the fluorescent signal of the nucleotide incorporated, and the base call is made according to the corresponding fluorescence dye.
The DNA sequencing is done on a chip that contains many ZMWs. Inside of each ZMWs, a single active DNA polymerase with a single molecule of single stranded DNA template is immobilized to the bottom through which the light comes through and creates a visualization chamber that allows monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a phospholinked 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 detecter to identify the base being incorporated by the DNA polymerase as it performs the
DNA synthesis. The fluorescent dye molecule is attached to a phosphate chain of the nucleotide. When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is cleaved off as the phosphate chain is cleaved off as a part of a natural DNA synthesisprocess when phosphodiester bondis created to elongate the DNA chain. The cleaved-off fluorescent dye molecule is then diffused out of the detection volume so that the fluorescent signal is not detected. [ [http://www.pacificbiosciences.com/assets/files/pacbio_technology_backgrounder.pdf Pacific Biosciences Technology Backgrounder] from [http://www.pacificbiosciences.com/index.php Pacific BiosciencesTM] ]
Zero-Mode Waveguide (ZMW)
The zero-mode waveguide is a nanophotonic confinment structure that consists of a circular hole in an aluminum cladding film deposited on the clear silica substrate [J. Korlach, P.J. Marks, R.L. Cicero, J.J. Gray, D.L. Murphy, D.B. Roitman, T.T. Pham, G.A. otto, M. Foquet, S.W. Turner, Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. PNAS. 105(2008) 1176-1181] . The ZMW holes are 70nm in diameter and 100nm in depth. Due to the behaviour of light when it travels through a small chamber, the optical field inside of the aluminum clad decays exponentially inside the chamber [M. Foquet, K.T. Samiee, X. Kong, B.P. Chauduri, P.M. Lundquist, S.W. Turner, J. Freudenthal, d.B. Roitman, Improved fabricatin of zero-mode waveguides for single-molecule detection. Journal of Applied Physics. 103 (2008) 034301-1-034301-9] . The volume of the illuminated observable chamber of a ZMW is 20 zeptoliters (20 X 10-21 liters). At this volume, the activity of DNA polymerase incorporating a single nucleotide can be detected.
Current Status of Sequencing Performance
The Pacific BiosciencesTM company expects to commercialize the SMRTTM sequencing in 2010 or 2011. The prototype of the SMRTTM chip contains 1000 ZMW holes that allow the parallelized DNA sequencing. Each of the ZMW holes produces approximately 1,500 bp(base pair)in read lengths at the speed of 10 bp per second.
The Single Molecule Real Time sequencing will be applicable for a broad range of genomics research, namely:
* "De novo" genome sequencing: The read length from the Single Molecule Real Time sequencing is currently comparable to that from the Sanger sequencing method based on dideoxynucleotide chain termination. The longer read length allows "de novo" genome sequencing and easier genome assemblies.
* Individual whole genome sequencing: Individual genome sequencing may utilize the Single Molecule Real Time sequencing method for the
* Resequencing: A same DNA molecule can be resequenced independently by creating the circular DNA template and utilizing a strand displacing enzyme that separates the newly synthesized DNA strand from the template. [ [http://www.pacificbiosciences.com/assets/files/pacbio_technology_backgrounder.pdf Pacific Biosciences Technology Backgrounder] from [http://www.pacificbiosciences.com/index.php Pacific BiosciencesTM] ]
* [http://www.pacificbiosciences.com/index.php Pacific BiosciencesTM company]
* [http://www.bio-itworld.com/BioIT_Content.aspx?id=71746 Report from the BioIT World.com]
* [http://www.genomeweb.com/issues/news/144976-1.html Report from Genome Web]
* [http://www.nytimes.com/2008/02/09/business/09genomebar.html?_r=1&ref=business&oref=slogin Report from New York Times]
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