分子生物学分子生物学 (15).pdf

Chapter 7Basic Methods of Gene Structure and Expression Analysis7.1 DNA sequence analysisDNA DNA sequence analysissequence analysis7.1.1 The dideoxy terminal termination method7.1.2 Automation of DNA sequence analysisFrederick Sanger1918 2013Walter Gilbert1932Establishment of DNA sequence analysis technologyThe Nobel Prize in Chemistry 19801.Dideoxy Chain TerminationDNA synthesis reactionDNA synthesis reactionStructureStructure ofof ATPATP,dATPdATP andand ddATPddATPAOHATPOHdATPAOHddATPAHTerminal termination81.1.Principle of dideoxy end Principle of dideoxy end termination methodtermination methodddNTPIn the DNA synthesis reaction,there is no ddNTPIn the DNA synthesis reaction,there is no 33-OH end of ddNTP,so it cannot form a 3,5OH end of ddNTP,so it cannot form a 3,5-phosphodiester bond with the 5phosphodiester bond with the 5-P end of the next P end of the next nucleotide,resulting in the extension of the new nucleotide,resulting in the extension of the new DNA strand to ddNTP early.DNA strand to ddNTP early.ddCTPddATPddTTPddGTPdCTPdATPdTTPdGTP5 T4711G A A T G A 3A C G C TDNA sequencing technology foundation:DNA sequencing technology foundation:HighHigh-resolution denatured resolution denatured polyacrylamide gel polyacrylamide gel electrophoresis(PAGE)electrophoresis(PAGE)technologytechnology.PAGE can separate singlePAGE can separate single-stranded oligonucleotide DNA stranded oligonucleotide DNA fragments with a length of fragments with a length of 300300-500 bases and a 500 bases and a difference of only 1 basedifference of only 1 base.A G C T5.Denatured polyacrylamide gel electrophoresis1.Preparation of single-stranded DNA template2.Annealing of DNA template and sequencing primers3.Incorporation labeling reaction4.Extension-stop reaction6.AutoradiographySequencing steps Sequencing steps 2 2.MainMain stepssteps ofof DNADNA sequencingsequencingKnown sequenceUnknown sequencedenatureTemplate-primer annealingTag reactionExtension-stop reactionSequencing primerSequencing primer extensionCCTAGGCTGGATCCGAGGATCCGAGGATCCGAReaction tube AGGATCCGAReaction tube GReaction tube CReaction tube TddATP&dNTPSddGTP&dNTPSddCTP&dNTPSddTTP&dNTPS53535335Single chain template5535Double-stranded DNA to be testedDenatured polyacrylamide gel electrophoresisDenatured polyacrylamide gel electrophoresisAutoradiographyAutoradiographyRead the analysis resultsRead the analysis resultsG反应管Reaction tube AReaction tube CReaction tube GReaction tube T13Known sequenceUnknown sequencedenatureTemplate-primer annealingTag reactionExtension-stop reactionSequencing primerSequencing primer extensionCCTAGGCTGGATCCGAGGATCCGAGGATCCGAReaction tube AGGATCCGAReaction tube GReaction tube CReaction tube TddATP&dNTPSddGTP&dNTPSddCTP&dNTPSddTTP&dNTPS53535335Single chain template5535Double-stranded DNA to be testedDenatured polyacrylamide gel electrophoresisDenatured polyacrylamide gel electrophoresisAutoradiographyAutoradiographyRead the analysis resultsRead the analysis resultsG A C TAutoradiographyAutoradiographyRead the analysis resultsRead the analysis resultsddCTP ddATPddTTPddGTPdCTPdATPdTTPdGTP5 TG A A T G A 3A C G C T2.Automation of DNA sequence analysis2.Automation of DNA sequence analysisddCTP ddATPddTTPddGTPdCTPdATPdTTPdGTP5 TG A A T G A 3A C G C T19Thank You!