现代生物科技课件

III.Chromosomereplicationiscoordinatedwithcelldivision2.Timingofinitiationofreplication(1)Thebacteriaaresynchronized,andthentheamountofDNAismeasured.(2)Fig.1.20showsthetimingofDNAreplicationduringthecellcycle,withtwodifferentgenerationtime.III.ChromosomereplicationiscoordinatedwithcelldivisionI,thetimefromreplicationinitiatedtoanewroundbegins.C,thetimeofentirechromosomereplication.(40at37oC)D,thetimefromreplicationcompletedtocelldivisionoccurs.(20at37oC)(1)C,Dremainthesameindependentofthegrowthrate.(2)Igetsshorterwhencellgrowthisfast,andcellhasshortgenerationtime(thetimeittakesanewborncelltogrowanddivides.(3)Theinitiationofchromosomereplicationoccurseachtimethecellachievesacertainmass,theinitiationmass.III.Chromosomereplicationiscoordinatedwithcelldivision4.ThetimingofinitiationofchromosomereplicationistiedtotheintracellularconcentrationofDnaAprotein.5.DammethylasecanmethylatetwoAsinGATC/CTAGsequence(1)Thissequencerepeated11timesin245bporiCregion.i.ImmediatelyafteranoriCregionhasbeenusedtoinitiatereplication,theGATC/CTAGsequencesinoriCarehemimethylated.ii.ThehemimethylatedoriCregionisseqesteredbybindingtothemembrane(mayhelpbyproteinSeqA),aprocessthatrendersitnonfunctionalfortheinitiationofnewroundsofreplication.iii.ThissequencealsoexistsinDanA promoter,andnoDnaAissynthesizedunlessthesesequencesarefullymethylated.(2)ThereisdirectevidencetosupporttheroleofmethylationinoriCsequestrationandregulationofDnaAproteinsynthesisafterinitiation.III.ChromosomereplicationiscoordinatedwithcelldivisionIV.SomemethodsforDNAstudyNucleicacidshybridizebybasepairingAcrucialpropertyofdoublehelixistheabilitytoseparatethetwoAcrucialpropertyofdoublehelixistheabilitytoseparatethetwostrandswithoutdisruptingcovalentbond.Thismakesitpossibleforstrandswithoutdisruptingcovalentbond.Thismakesitpossibleforthestrandstoseparateandreformunderphysiologicalconditionsatthethestrandstoseparateandreformunderphysiologicalconditionsatthe(veryrapid)ratesneededtosustaingeneticfunctions.(veryrapid)ratesneededtosustaingeneticfunctions.Heatingandsomechemicals(e.g.,NaOH)causethetwostrandsofaDNAHeatingandsomechemicals(e.g.,NaOH)causethetwostrandsofaDNAduplextoseparate.duplextoseparate.ComplementarysinglestrandscanrenaturewhenthetemperatureisComplementarysinglestrandscanrenaturewhenthetemperatureisreduced.reduced.Denaturationandrenaturation/hybridizationcanoccurwithDNA-DNA,Denaturationandrenaturation/hybridizationcanoccurwithDNA-DNA,DNA-RNA,orRNA-RNAcombinations,andcanbeintermolecularorDNA-RNA,orRNA-RNAcombinations,andcanbeintermolecularorintramolecular.intramolecular.TheTheT Tmmisthemidpointofthetemperaturerangefordenaturation.isthemidpointofthetemperaturerangefordenaturation.Theabilityoftwosingle-strandednucleicacidpreparationsTheabilityoftwosingle-strandednucleicacidpreparationstohybridizeisameasureoftheircomplementarity.tohybridizeisameasureoftheircomplementarity.DNAMeltingTheamountofstrandseparation,ormelting,ismeasuredbytheabsorbanceofDNAsolutionat260nmGCcontentofDNAhasasignificanteffectonTmwithhigherGCcontentmeaninghigherTmnoncovalentforceshydrophobicpropertyofbases,hydrophilicpropertyofbackboneandhydrogenbonds Withheating,noncovalentforcesholdingDNAstrandstogetherweakenandbreakWhentheforcesbreak,thetwostrandscomeapartindenaturationormeltingTemperatureatwhichDNAstrandsaredenaturedisthemeltingtemperatureorTmDenaturedsinglestrandsofDNAcanrenaturetogivetheduplexform.FilterhybrydizationCalculationofTmforaDNAmolecule1.Tm=(G+C)%/2.44+81.5+16.6logNa+2.For oligonucleotide:Tm=4(G+C)+3(A+T)3.In the presence of foramide:minus 0.67 0C per%4.High stringency and low stringencyMeasurementofnucleicacidconcentration1.Hypochromic effect:The heterocyclic rings of 1.Hypochromic effect:The heterocyclic rings of nucleotides absorb light strongly in UV range(with nucleotides absorb light strongly in UV range(with maximum close to 260 nm that is characteristic for each base).maximum close to 260 nm that is characteristic for each base).But the absorption of DNA itself is 40%less than would be But the absorption of DNA itself is 40%less than would be displayed by a mixture of free nucleotides of the same displayed by a mixture of free nucleotides of the same position.2.Concentration of nucleic acid in a solution(ug/ml):2.Concentration of nucleic acid in a solution(ug/ml):DNA:OD DNA:OD260260 X 50 X(dilution factor)X 50 X(dilution factor)RNA:OD RNA:OD260260 X 40 X(dilution factor)X 40 X(dilution factor)Oligo:OD Oligo:OD260260 X 30 X(dilution factor)X 30 X(dilution factor)RestrictionenzymesRestrictionandmodification(限制與修飾限制與修飾)RestrictionenzymesA.CleaveDNAatspecificsequences(recognitionA.CleaveDNAatspecificsequences(recognitionsequence,cuttingsite).sequence,cuttingsite).(一種可以針對特定一種可以針對特定一種可以針對特定一種可以針對特定DNADNA序列進行辨認與切割的蛋白質序列進行辨認與切割的蛋白質序列進行辨認與切割的蛋白質序列進行辨認與切割的蛋白質)B.FirstlydiscoveredbyArberandSmith(late1960s)B.FirstlydiscoveredbyArberandSmith(late1960s)C.Boyer(1969)firstisolatedC.Boyer(1969)firstisolatedEcoEcoRIRID.DifferentrestrictionenzymesmaysharethesameD.Differentrestrictionenzymesmaysharethesamerecognitionsequencealthoughtheydonotnecessaryrecognitionsequencealthoughtheydonotnecessarycutatpreciselythesameplace(isoschizomers,cutatpreciselythesameplace(isoschizomers,同切同切同切同切 點酶點酶點酶點酶).).E.There are two major classes of restriction enzyme that differ in E.There are two major classes of restriction enzyme that differ in E.There are two major classes of restriction enzyme that differ in E.There are two major classes of restriction enzyme that differ in where they cut the DNA,relative to the recognition site.where they cut the DNA,relative to the recognition site.where they cut the DNA,relative to the recognition site.where they cut the DNA,relative to the recognition site.1.Type I restriction enzymes cut the DNA a long way from the 1.Type I restriction enzymes cut the DNA a long way from the 1.Type I restriction enzymes cut the DNA a long way from the 1.Type I restriction enzymes cut the DNA a long way from the recognition sequence.recognition sequence.recognition sequence.recognition sequence.2.Type II restriction enzymes cut the DNA within the recognition 2.Type II restriction enzymes cut the DNA within the recognition 2.Type II restriction enzymes cut the DNA within the recognition 2.Type II restriction enzymes cut the DNA within the recognition sequence.Some generate blunt ends,others give sticky ends.sequence.Some generate blunt ends,others give sticky ends.sequence.Some generate blunt ends,others give sticky ends.sequence.Some generate blunt ends,others give sticky ends.RestrictionfragmentendsSeparationofDNAfragmentsbygelelectrophoresisThere are kinds of gels for electrophoresis:A.agarose（瓊脂糖）and polyacrylamide（聚丙烯醯胺）gel electrophoresis (膠体電泳)B.Agarose and polyacrylamide can act as a molecular sieve C.Agarose is better for separation of DNA fragments 1 kb,and polyacrylamide gel for 1kb.ElectrophoresiscanseparateDNAfragmentsfromoneanother（電泳可將（電泳可將DNA片段彼此分開）片段彼此分開）DNA fragments are separated using an electrical DNA fragments are separated using an electrical current:current:a.Apparatus and material a.Apparatus and material casting tray,comb,casting tray,comb,loading dye,ethidium bromide loading dye,ethidium bromide（溴溴化乙錠）化乙錠）化乙錠）化乙錠）and and power supply power supply b.DNA molecules have a negative electric charge b.DNA molecules have a negative electric charge due to the phosphate groups which alternate due to the phosphate groups which alternate with sugar molecules to make up the backbone with sugar molecules to make up the backbone of the DNA double helix.of the DNA double helix.c.c.Opposite electric charges tend to attract one Opposite electric charges tend to attract one another.another.Horizontalapparatusfor(agarose)gelelectrophoresismovement ofDNA in electricalfieldGel electrophoresisGelelectrophoresisforDNAfragmentsPulsed-FieldGelElectrophoresis(PFGE)當兩個電磁場以規則的方式相互交替，DNA分子在膠體中之淨值移動仍然是從一端到另外一端，移動方向或多或少還是直線的。然而，隨著每一個電磁場方向的改變，每一DNA分子在繼續往前移動之前必須作90度重新排列。這就是這種技術的重點，因短的分子重新調整比長的分子快，使得短的分子朝膠體尾端前進的速度快。這種新增的空間戲劇性地增大了膠體的解析力，因此大至好幾千kb 長的分子可以被分開。SouthernblotanalysisPCRreaction(聚合酵素鏈鎖反應)PCRreaction(聚合酵素鏈鎖反應)The Determining base sequence of DNA fragments（定序列）（定序列）A.A.Maxam and Gilbert DNA sequencingMaxam and Gilbert DNA sequencing a.a.base modification chemicals:base modification chemicals:1.DMS(dimethylsulfate)1.DMS(dimethylsulfate)G G 2.DMS in the presence of formic acid 2.DMS in the presence of formic acid G and A G and A 3.HZ(hydrazine)3.HZ(hydrazine)T and C T and C 4.HZ in the presence of NaCl 4.HZ in the presence of NaCl C C b.cleavage of modified base-piperidine b.cleavage of modified base-piperidineB.Sanger dideoxy-DNA sequencingB.Sanger dideoxy-DNA sequencing a.a.dideoxy dNTPdideoxy dNTP b.b.primersprimersC.C.Denaturing polyacrylamide sequencing gelDenaturing polyacrylamide sequencing gelD.AutoradiographyD.AutoradiographyE.AutomatedsequencingE.AutomatedsequencingMaxam&GilbertSequencingStructureofan-35S-deoxynucleotidetriphosphateDideoxynucleosidetriphosphates(ddNTP)DNAsequencingwithddNTPsaschainterminatorsProfileofanautomatedsequencingLabellingtheProbesNick translationNick translationRandom primimgRandom primimg3-end-labelling a DNA by end-filling3-end-labelling a DNA by end-fillingEnd-labelling by polynucleotide kinaseEnd-labelling by polynucleotide kinaseFig.5.293-end-labellingaDNAbyend-filling