Single molecule real time sequencing
SMRT sequencing based on sequencing-by-synthesis approach. For capturing an instrument located at the bottom of the well, the container and similar small – DNA was synthesized zero-mode waveguide (ZMWs). To give a free solution, the array is performed using nucleotides fluorescently labeled with (attached to it ZMW) unmodified polymerase. Well is designed only fluorescence generated in the well bottom so open. It is separated from the nucleotides by leaving the DNA strand that is not modified with fluorescent labels, to incorporate them into DNA strands. According Biosciences, Pacific Ocean, the SMRT technology developer, this methodology can be used to detect (such as cytosine methylation, for example) nucleotide changes. This occurs on the observation polymerase dynamics in. This approach makes it possible reading of 20,000 or more nucleotides, the average length of 5 km reading-based.
The synthesis technique has been developed (also known as SMRT) by the Pacific Ocean Biosciences Inc., is parallelized a single molecule DNA sequencing single molecule real-time sequence. Use zero-mode wave, which was developed in the laboratory of Harold G. and (ZMW), Watt W. Webb of Cornell University, and single-molecule real-time sequencing Craighead. The enzyme, DNA polymerase is located at the bottom of the ZMW the DNA molecule as a template. Is a DNA polymerase having the structure illuminated observation volume small enough is created to observe only one nucleotide (also known as base) in the DNA ZMW. Each of the 4 DNA bases are attached to one of the different fluorescent dyes four. If it is incorporated by DNA polymerase, cut a fluorescent marker nucleotides are expanded surveillance area ZMW whose fluorescence is not observed. Detecting the fluorescent signal of the nucleotide incorporation, the base, the detector is a call by fluorescence corresponding dyes. Sequence data generated from a single molecule real time sequencing, was published in January 2009 in the journal Science first.
The DNA sequencing, I went a chip that contains the ZMWs many. In each ZMW, through may be created through the imaging camera capable of light to track the activity of DNA polymerases at the molecular level, DNA polymerase and a single single-stranded DNA template molecule, the bottom is fixed. Signal from the incorporated nucleotide phosphate binders is detected go to DNA synthesis, as results in DNA sequencing in real time by a DNA polymerase. The nucleotide bases each detector has a fluorescent dye for each of the four in order to allows to determine on the basis of the from being taken by the DNA polymerase DNA synthesis to occur. And is attached to the phosphate of the nucleotide chains of the fluorescent dye molecule. Nucleotides, when it is incorporated by DNA polymerase, when establishing a phosphodiester bond to the DNA chain elongation, as part of the natural process of DNA synthesis, the fluorescent dye is split and a phosphoric acid chains. Fluorescent dye molecules which are separated and then the fluorescent signal becomes not detected, the spray from the detection of sound. It is a nano-photonic birth structure is composed of a circular hole in the lining of aluminum foil silica was deposited (ZMW) on a clear substrate in wave mode of zero. In diameter ~ 70nm in ZMW hole, there is about 100 Nm depth. It is due to the behavior of light passing through the small hole of the optical field profile decays exponentially into the chamber. ~ 20 zeptoliters observables in ZMW is illuminated (10-21 × 20 liters). This volume inside, it is possible to easily detect the activity of DNA polymerase for single nucleotide.
In other words, real-time single molecule sequence to be applied to a variety of genomic research. Please refer to the length of the molecule from a single real-time sequence greater than that of the method sequence based on the Sanger dideoxy chain termination or equivalent: genome sequence new de. I want to enable the genome assembly and easy de novo of long read length genome. The SMRT sequencing, in the New England Journal of Medicine, in 2011, publication of Denobogenomu was demonstrated for analyzing the occurrence of E. coli in Germany in the cholera epidemic in Haiti in 2010. The scientists also, in order to balance the short read sequence data and read sequence data long-term, I use a single molecule real-time sequence of de novo genome hybrid meeting. In 2012, publication of peer review of some proven, the genome of complete bacteria that are automated including a newspaper that updates the assembler Celera genome finish pipe using the SMRT sequencing long read was released.
DNA molecules, and using an enzyme-shift circuit for separating the DNA strand newly synthesized from the template, it is possible to create a circular DNA template, and re-sequencing is independently: rearranged. SMRT is other Pacific Ocean Biosciences, using this approach round consensus with the sequencing to prove the validity of the activating internal tandem duplication mutation in FLT3 as a therapeutic target in acute myeloid leukemia, and the University of California scientists from institutions. These results, it is published in Nature magazine in April 2012. August 2012, scientists of the Broad Institute, announced the evaluation of SNP calls for SMRT sequencing. Methylation Detection: to indicate base whether it is methylated, and the dynamics of the polymerase. In order to detect changes and other methylation based on peer-reviewed articles in a series in 2011, scientists, shows the use of real-time sequencing of single molecules. In 2012, for the team of scientists SMRT used to generate a full methylomes, sequencing the six bacteria said the findings in the study of nucleic acids. November 2012, scientists have published a report on the methylation of genome-wide epidemic strain of E. coli.