第九章DNA序列总结.ppt

一、Maxam-Gilbert 化学降解法测序技术 DNA sequencing by chemical degradation G 反 应： 硫酸二甲脂DMS使G鸟嘌呤的7位氮原子甲基化，其后断开第8位碳原子和第9位氮原子间的化学键，哌啶置换了被修饰鸟嘌呤与核糖的结合。 G+A反应： 甲酸使嘌呤环上的氮原子质子化，削弱了腺嘌呤脱氧核糖核苷酸和鸟嘌呤脱氧核糖核苷酸中的糖苷键，然后哌啶置换了嘌呤。 T+C反应： 肼断开了嘧啶环，产生的碱基片段能被哌啶所置换。 C 反 应： 在NaCl存在时，只有C才能与肼发生反应，随后被修饰的胞嘧啶被哌啶置换。 下面的一个点就代表一个簇！ Illumina / Solexa: Genetic Analyzer Real Time Sequencing by Synthesis Clonal Single Molecule Array Amplification: bridging PCR 60 million reads / run up to 50 bases / read 2 Gb / run 8 channels, app. 5 mio reads / channel Fluorescent labels Reversible 3‘OH blocking Illumina / Solexa Genetic Analyzer 2000 Mb / run Real Time Sequencing by Ligation Emulsion PCR and Beads on slides 85 million reads / run Up to 35 bases/read 3 Gb / run dual fluorescent labels 8 individual channels / flowcell 2 flowcells / run Applied Biosystems SOLiD 3000 Mb / run Oligonucleotide Ligation Detection (SOLiD) Comparison of “Next Generation” Sequencing Technologies 每一组样品所给出的条带数目有限， 由此读出的核苷酸序列也有限 Lane A: dNTP + ddATP Lane C: dNTP + ddCTP Lane G: dNTP + ddGTP Lane T: dNTP + ddTTP 2 loading (2) PCR reaction (2) PCR reaction CircumVent? Thermal Cycle Dideoxy DNA Sequencing Kit with Vent R? (exo-) DNA Polymerase Brief protocols Denature templates (ssDNA or ds DNA) 65 ?C to 35 ?C 15~30min Annealing Labeling RT 2~5min Termination 37?C 5min, stop solution Running gel 75?C 2min, running at 55 ?C, 2 time of loading 100?C 2min P C R PCR procedure 95 ?C 20 sec 55 ?C 20 sec 72 ?C 20 sec 20 cycles T T T A A G T G A A A A A A C A G C T* T A A A T T C C A A A A G C A T T A T A G A A G T* A T C A A T T T T G C A Primer extension for CTC gene (3) Automatic sequencing ABI PRISM? dRhodamine Terminator Cycle Sequencing Ready Reaction Kit With AmpliTaq? DNA Polymerase, FS PE Applied Biosystems a division of Perkin-Elmer Core principle Key technique Labeling ddNTP with 4 kind of dRhodamine Each dRhodamine give different color A green C yellow G bl