User:ElizChun/draftSMRT

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Single Molecule Real Time Sequencing (also known as SMRTTM) is a paralleled single molecule DNA sequencing by synthesis developed by Pacific BiosciencesTM. Pacific Biosciences is based in Menlo Park, California, founded in 2004. The Single Molecule Real Time sequencing is based on the usage of Zero-Mode Waveguide (ZMW), developed in the laboratory of Dr. Harold G. Craighead [1] at Cornell University. A DNA polymerase is affixed at the bottom of the 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 smally enough to observe only a single nucleotide being incorporated by DNA polymerase. Each of the four bases of DNA molecules is attached to four different fluorescent dye at the phosphate chane of the nucleotide. When a nucleotide is incorporated into the double-stranded DNA chain, 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.

Contents


[edit] Technology

Overview of Single Molecule Real Time Sequencing
Overview of Single Molecule Real Time Sequencing

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.


[edit] Nucleotide phospholinkage

For each of the nucleotide base, there are four corresponding fluorescent dye molecules that enable the detecter to identify the base being incorporated by the DNA polymerase as it synthesizes the double stranded DNA. The fluorescent dye molecule is attachd 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 synthesis process. The cleaved off fluorescent dye molecule is then diffused out of the detection volume so that the fluorescent signal is not detected. [2]


[edit] Zero-Mode Waveguide (ZMW)

The Zero-mode waveguide is a nanophotonic confinment structure that consist of a circular hole in an aluminum cladding film deposited on the clear silica substrate[3]. 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 [4] . The size of the ZMW creates the illuminated observable chamber at the volume of 20 zeptoliters (20 X 10-21 liters). At this volume, the activity of DNA polymerase incorporating a single nucleotide can be detected.

[edit] Current status of sequencing performance

The Pacific Biosciences company expects to commercialize the SMRTTM sequencer in 2010 or 2011. The prototype SMRT chip contains 1000 ZMW holes that allows for the parallelized sequencing, each of which produces ~1,500 bp in read lengths at the speed of 10bp per second.

[edit] Application

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 dideoxynucleotide chain termination. The longer read length allows for a de novo genome sequencing and an easier genome assembly.
  • Individual whole genome sequencing: Individual genome sequencing may utilize the Single Molecule Real Time sequencing for the personalized medcine
  • 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[5].

[edit] External links

[edit] Refernces

  1. ^ 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
  2. ^ Pacific Biosciences Technology Backgrounder from Pacific BiosciencesTM
  3. ^ 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
  4. ^ 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
  5. ^ Pacific Biosciences Technology Backgrounder from Pacific BiosciencesTM
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