SMRT DNA Sequencing

Pacific biosciences is developing a transformed DNA sequencing technology, which will revolutionize the field of genetic analysis by enabling researchers to answer questions important to human healthcare, It is called SMRT (Single Molecule Real-Time DNA Sequencing). A breakthrough technology based on the natural process that occurs every time living cells divides.

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Prior to division DNA is replicated by enzymes called DNA polymerase is which efficiently duplicating entire genomes in minutes, by reading the DNA and sequentially building a complementary strand with matching building blocks called nucleotides. Pacific biosciences SMRT sequencing harnesses the power of the polymerase as sequencing engine by eavesdropping on what works to replicate DNA, this approach is enabled by two proprietary technologies the first Phospholinked nucleotides. To visualize polymerase activity a different colored fluorescent label is attached to each of the four nucleotides ACG and T. In contrast to other sequencing approaches R phosholinked nucleotides carry their fluorescent label on the terminal phosphate rather than the base. Through this innovation enzyme cleaves away the fluorescent label as part of the incorporation process leaving behind a completely natural strand of DNA.This enables us to exploit the inherent properties of the DNA polymerase including high-speed long read length and high fidelity. The second key technology is a nano photonic visualization chamber called the Zero Mode Wave-guide or ZMW, It enables observation of the individual molecules against a required background of labeled nucleotides while maintaining a high signal-to-noise. This ZNW is a cylindrical metallic chamber approximately 70 nm wide it is illuminated/support creating an extremely small detection volume just 20 cL. Nucleotides defused in and out of the ZNW in microsecond. When the polymerase encounters the correct nucleotide it takes several milliseconds to incorporated during which time its florescent label is excited emitting light is captured by a sensitive detector. After incorporation the label is clipped off and diffuses away, The whole process repeats creating sequential bursts of light corresponding to the different nucleotides these are recorded thus building the DNA sequence.

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 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 personalized medicine.
  • 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.

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