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最新的基因测序技术ABI的SoLiD系统
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Next generation sequencing technology comprises clonal cluster and single molecule sequencing. Following is a brief overview of both technologies and their multiple methodologies. Clonal cluster sequencing All next generation sequencing systems, including the Applied Biosystems SOLiD System, use clonal cluster sequencing. The process, which begins with a single target molecule, involves creation of a clonal target during an intermediate amplification step. Multiple identical copies are required to produce a high signal-to-noise-ratio. Single molecule sequencing True single molecule sequencing uses a single molecule as the unit of measurement and requires no amplification. Clonal Cluster Methodologies Clonal cluster sequencing includes two methodologies: sequencing by synthesis (SBS) and sequencing by ligation (SBS). Sequencing by Synthesis (SBS) Sequencing by synthesis generates a DNA sequence by taking a measurement as each base is added. SBS uses both terminator and pyrosequencing chemistries. Terminal Chemistry In terminator chemistry, all four fluorescently labeled nucleotides are added, and each competes for incorporation. After incorporation of a single base, a fluorescent dye measures the incorporated nucleotide. The dye must be removed before the next base can be incorporated. Pyrosequencing Chemistry During pyrosequencing chemistry, every base is added sequentially. There is no competition, and the signal, which is recorded for a single base, is proportionate to the number of bases present. The signal strength for TTT, for example, would be expected to be three times stronger than the signal strength for T. On average, ~1.5 cycles are required to measure each base. Every cycle begins with the addition of deoxynucleotide triphosphates (dNTP), followed by measurement of the signal, which is created by the chemiluminescence (sulfyrase and luciferase) that is released upon incorporation. The cycle is repeated for the remaining three deoxynucleotide triphosphates (dNTP). Sequencing by Ligation (SBL) Sequencing by ligation, the technology used in the Applied Biosystems SOLiD? System, generates DNA by measuring the serial ligation of an oligonucleotide. All fluorescently labeled oligonucleotide probes are present simultaneously and compete for incorporation. After each ligation, the fluorescence signal is measured and then cleaved before another round of ligation takes place. A reset phase allows a reduction in noise—a capping step that prevents dephasing. |
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