第二代测序原理及过程.pptVIP

The Roche/454 FLX /enablingtechnology/the-system.asp The Illumina/ Solexa Genome Analyzer /pages.ilmn?ID=203 The Applied Biosystems SOLiDTM /images/Product/Solid Knowledge/flash/102207/solid.html 第二代测序流程 Menu 咖啡地图 收拾心情 细水长流 有关文化 Next-Generation DNASequencing Methods The method used by the Roche/454 sequencer to amplify single-stranded DNA copies from a fragment library on agarose beads. A mixture of DNA fragments with agarose beads containing complementary oligonucleotides to the adapters at the fragment ends are mixed in an approximately 1:1 ratio. The mixture is encapsulated by vigorous vortexing into aqueous micelles that contain PCR reactants surrounded by oil, and pipetted into a 96-well microtiter plate for PCR amplification. The resulting beads are decorated with approximately 1 million copies of the original single-stranded fragment, which provides sufficient signal strength during the pyrosequencing reaction that follows to detect and record nucleotide incorporation events. sstDNA, single-stranded template DNA. The sequencing instrument consists of the following major subsystems: a fluidic assembly (A) a flow chamber that includes the well-containing fibre optic slide (B) a CCD camera-based imaging assembly (C) a computer that provides the necessary user interface and instrument control. 上次问题 Typical histogram of signal intensities for negative and positive flows The Illumina sequencing-by-synthesis approach. Cluster strands created by bridge amplification are primed and all four fluorescently labeled, 3-OH blocked nucleotides are added to the flow cell with DNA polymerase. The cluster strands are extended by one nucleotide. Following the incorporation step, the unused nucleotides and DNA polymerase molecules are washed away, a scan buffer is added to the flow cell, and the optics system scans each lane of the flow cell by imaging units called tiles. Once imaging is completed, chemicals that effect cleavage of the fluorescent labels and the 3-OH bloc