测序技术原理介绍ppt课件.ppt
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1、测序技术原理介绍,化学测序Sanger测序NGS(二代测序)Form the next to the third(三代测序),测序方法分类,手工测序自动测序,Sanger双脱氧链终止法,Maxam-Gilbert化学降解法,3100/3100-a,3130/3130XL,3700,3730/3730XL,Solexa,454,焦磷酸法,SOLiD,Gilbert化学降解法1. 目的DNA制备2. 单侧末端标记待测DNA片段3. 碱基的特异性修饰及化学降解4. 聚丙烯酰胺凝胶电泳5. 放射自显影6. 阅读测序结果,Sanger测序法1. 单链DNA模板制备2. DNA模板与测序引物退火3. 掺入
2、法标记反应4. 延伸终止反应5. 变性聚丙烯酰胺凝胶电泳6. 放射自显影7. 阅读测序结果,Chain-terminator reaction,deoxynucleotides, dNTP,dideoxynucleotides, ddNTP,Frederick Sanger,Original Sanger sequencing,1980-1990Radio gelRead length: 102bp 103 bp / day,Automated Sanger sequencing,1990-Fluorescent capillaryRead length: 103bp106bp / day,A
3、BI 3730 DNA Analyzer,$376K/instrument650 bp/read0.06 Mb/run2h/run$10K/Mb,Hierarchical shotgun (map-based) method,Long fragment,BAC library,Physical map,Shotgun,Sequencing and assembly,Human Genome Project$3 billion, 11 years,Whole-genome shotgun method,Short fragment,Plasmid library,Mate-pair sequen
4、cing,3 years$300 million,Craig Venter,Sanger vs next-generation sequencing,Nat Biotech 26:1135(2008),Roche 454,Since 2004,Genome Sequencer / FLX,Template preparation- emulsion PCR,Trends in Genet 24:133(2008),Pyrosequencing,Single dNTP type flows per cycleInorganic pyrophosphate (PPi) drives visible
5、 light through a series of reactionsRemove unincorporated nucleotide,Trends in Genet 24:133(2008),Base calling,Homopolymer error,GV6330,Illumina Solexa,Since 2006,Genome Analyzer,Template preparation-bridge RCR,Adaptor ligation,Surface attachment,Bridge amplification,Denaturation,Trends in Genet 24:
6、133(2008),Cyclic reversible termination,All four labeled reversible terminators are added per cycleRemove unincorporated bases and detect signalRemove the terminating group and the fluorescent dye,Trends in Genet 24:133(2008),Terminating group,Fluorophore cleavage,Nat Rev Genet 11:31(2010),Base call
7、ing,Solexa测序原理,Main Illumina noise factors,(a) In the ideal situation, after several cycles the signal (green arrows) is strong, coherent and corresponds to the interrogated position.(b) Phasing noise introduces lagging (blue arrows) and leading (red arrow) nascent strands, which transmit a mixture
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