DNA Sequencers

DNA sequencers have become more important due to large genomics projects and the need to increase productivity.

Modern automated DNA sequencing instruments (called DNA sequencers) are able to sequence as many as 384 fluoresecently labeled samples in a batch (run) and perform as many as 24 runs a day. These perform only the size separation and peak reading; the actual sequencing reaction(s), cleanup and resuspension in a suitable buffer must be performed separately.


The magnitude of the fluorescent signal is related to the number of strands of DNA that are in the reaction. If the initial amount of DNA is small, the signals will be weak. However, the properties of PCR allow one to increase the signal by increasing the number of cycles in the PCR program.


A simple DNA sequencer will have one or more lasers that emit at a wavelength that is absorbed by the fluorescent dye that has been attached to the DNA strand of interest. It will then have one or more optical detectors that can detect at the wavelength that the dye fluoresces at. The presence or absence of a strand of DNA is then detected by monitoring the output of the detector. Since shorter strands of DNA move through the gel matrix faster they are detected sooner and there is then a direct correlation between length of DNA strand and time at the detector. This relationship is then used to determine the actual DNA sequence.










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Cycle Sequencing

To sequence a piece of dna you need 1)a Template DNA 2) a short DNA primer that is complementary to the dna you want to sequence, 3)A enzyme called DNA polymerase,(4) Four nucleotides.(A,C,G,T), To this mix ,we also add a second type of nucleotide; one that has a slightly different chemical formula, These dideoxynucleotides(diddtp) can be recognized by a DNA sequencer.

To start the sequencing reaction this mixture is heated to 96C ,so the template DNA's two complementary strand separates,Then the temperature is lowered, so that the short "primer" sequence finds its complementary sequence in the template DNA.Finally the temperature is raised 60c,this allows the enzyme to bind to the DNA and create a new strand of DNA.





The sequence of this new DNA is complementary to the original DNA strand. The enzyme makes no distinction between dNTPs or didNTPs.each time a didNTP is incorporated, in this case didATP,The synthesis stops. Because billion of DNA molecules are present in the test tube, the strand can be terminated at any position. This results in collection of DNA strands of many different lengths.


The sequencing reaction is transferred from the test tube to a lane of a polyacrylamide gel. The gel is placed into a DNA sequencer for electrophoresis and analysis. The fragments migrate according to size and each is detected as it passes a laser beam at the bottom of the gel. Each type of dideoxynucleotide emits colored light of a characteristic wavelength and is recorded as a colored band on a simulated gel image, and finally computer program interprets the raw data and outputs an electropherogram with colored peaks representing each letter in the sequence.the sequence fragments are sorted out according to the size, starting from the shortest to longest one, the stimulated gel image is read from bottom to top, starting with the smallest fragment, Thus we sequences present in template DNA.

















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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.







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.