Using Sequencing In The Study Of Dna Biology Essay

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Sequencing: Sequencing is the study of the order of nucleotides in the DNA/RNA sample. Its understanding is crucial is in living organism pathophysiology. (1, 2, 3)

Sanger and Coulson developed the chain termination method of DNA sequencing over 30 years ago. This method is based on using a modified deoxynucleotides called dideoxynucleotides ( ddCTP, ddGTP, or ddTTP ddATP) which act as chain-terminating nucleotides. they lack a hydroxyl group required for the formation of a phosphodiester bond between two nucleotides. DNA polymerase adds nucleotides to the 3' end of the newly-forming strand. Because of the different in the chemical structure in ddNTP, they cause chain termination variation in the length of the synthesized DNA fragments which can be radioactively labelled or fluorescent labelled and sequence can separate into the four different DNA fragment bands by gel electrophoresis or the fluorescence detected.(1)

Automated Sanger sequencing was used to perform the Human Genome Project which costs over 3 billion and took more than 10 years. Sanger method of DNA sequencing has downsides: the high cost and maximum read length of <1000. In addition to long experiment setup and large DNA concentration needed to run this process. These made biologist demand for low-cost sequencing which ultimately driven the development of high-throughput next-generation sequencing technologies. Roche was the first in the development of the next generation sequencing. The concept of next-generation sequencing or sequencing by synthesis relies on arraying thousands or millions of sequences at once to be analysed in parallel. High-throughput sequencing technologies are therefore intended to lower the cost of DNA sequencing and increasing sequencing throughput. (1) (4)

Recent scientific discoveries that resulted from the application of next generation DNA sequencing technologies highlight the striking impact of these massively parallel platforms on biology and medicine. Next-generation sequencers available commercially are; Roche (454-GS FLX), Illumina (Solexa) and Applied Biosystem (solid system). They all share the principle of using adapter ligated fragment library.(1) The application of next-generation sequencing has expanded the world of genomics. The sequencing of RNA also has transitioned and now includes full-length cDNA analyses and lately direct RNA sequencing. (5) Next-generation sequencing has also enabled sequencing of extintint animals’ DNA samples, and has substantially widened the scope of metagenomic. Taken together, an astounding potential exists for these technologies to bring enormous change in genetic and biological research and to enhance our fundamental biological knowledge. (1, 2, 3)

In the Roche/454 approach (pyrosequncing), DNA fragments needed to be sequenced are mixed with beads. These beads contain multiple nucleotides binding site but only one complementary nucleotide is attached. DNA fragment binds to the complementary nucleotide attached to the surface of the bead. The fragment: nucleotide bead complex is then separated from the rest of other beads by emulsion (micelle formation). The beads complex also contains PCR material, which upon activation of it results in DNA amplification. The amplified monoclonal fragments are then sequenced. DNA polymerase results in pyrophosphate release when a nucleotide gets incorporated. Pyrophosphate release causes downstream enzymatic reactions ultimately leading to release of light by luciferase. The amount of light produced from pyrophosphate formation are recorded by a camera and the amount of light produced is proportional to number of nucleotide incorporated. Few downsides to the Roche/454 sequencer are camera detector saturation from long reads of the same nucleotide (homopolymer) and hence error formation. (1)

The Illumina system uses a sequencing by-synthesis approach in which all four nucleotides, which are fluorescent labelled and carries a blocking group to make it unique, are added simultaneously to the flow cell channels, along with DNA polymerase, for incorporation into the oligo-primed cluster. Each nucleotide carries a blocking group which makes it unique for incorporation. An imaging step follows each base incorporation step. After each imaging step, the blocking group is removed and the process repeated. (1)

The third type of next generation sequencing device is Applied Biosystems. This device share the same principle of using adapter fragment library like the other two systems, and it shares usage of PCR similar to Roche 454, but unlike other next-generation platforms, it used DNA ligase rather than polymerase. The DNA fragments are then inserted into glass slide. (1)

Two key drawbacks in the next-generation sequencing are (a) shorter reading length compared to automated Sanger sequencer and (b) the cost especially the need for highly advanced computational analyses in addition to the chemical needed.(4)

The ever great demand to sequence the whole genome of human in a price around the region of $1000, has paved the way for Next (next) generation or third generation sequencing technology. The three currently discussed platforms in the scientific fields are: Helicos and Pacific Biosciences which detects fluorescently labelled nucleotides to perform single-molecule sequencing. The latest third generation sequencer is the Oxford Nanopore. (1, 4, 5, 7, 6, 8)

Single molecule real time sequencing is a parallelized single molecule DNA sequencing by synthesis technology developed by Pacific Biosciences. it uses zero-mode waveguide (ZMW). DNA polymerase enzyme is located at the bottom of a ZMW with a single molecule of DNA as a template. the DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW. A device detects the fluorescent signal of the nucleotide incorporation and sequenced. (4)

Helicos Genetic Analysis system uses Single Molecule Sequencing (SMS). The advantage of this platform is that Sample preparation does not require ligation or PCR amplification.DNA needs to be fragmented then attached to poly (A), and hybridized to a flow cell surface containing oligo (dt) for sequencing-by-synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. (7, 8)

Gene expression measurements can be done using first-strand cDNA-based methods or using a novel approach that allows direct hybridization and sequencing of cellular RNA for the most direct quantification possible. (5)

The Oxford Nanopore design uses the protien nanopore from α-hemolysin with the molecular adapter cyclodextrin covalently attached to the inside of the pore. An exonuclease digests single-stranded DNA, and as single bases fall into the pore, they transiently interact with the cyclodextrin and block an electrical current that runs through the pore. The current amplitude-characteristic for the individual bases A, G, C and T as well as methylcytosine-is then easily converted to DNA sequence. Each base has a characteristic mean dwell time in the millisecond range, its dissociation rate constant is voltage-dependent and a potential of +180 mV ensures that the base is swept out of the pore on the other side. (8)

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Nanopore sequencing has the advantage that it does not require any labelling of the DNA or fluorescent use. Also cameras to record from optical chips are not needed. This technology claims 98% reading accuracy and long reads; however it is early to predict the impact it will have on cheap whole-genome sequencing as parallelization is a technical issue. (8)