生科院学长分子生物学上

1、MolecularBiology: the studyof gene and theiractivitiesat the molecular level, it grewout of disciplinesofGeneticsandMendelslawsofEverycellcontainedMolecularBiology: the studyof gene and theiractivitiesat the molecular level, it grewout of disciplinesofGeneticsandMendelslawsofEverycellcontainedpairsc

2、torsteachpairdetermineda specifictrait(特点Lawofsegregation（分离The members of each pair segregated from each agametecontainedonememberofeachhe sof sex-cell formation, LawofindependentThesegregationofeachpairwasindependentofthesegregationofairsGeneisinMan explained the separation of certain inherited t

3、are usually linked causedbythebreakingofchromosomessometimesduringthesofcell . EachgeneisoanenzymetoperformtaskswithinDNA as genetic materialDNA structureRestriction PCRtechnologyTransgenicanimalandplantsGenomebinant DNA Gene& Innon-molecularterms,geneisaunitofternsthecharacterofaparticularoleculart

4、erms,asegmentofDNAcontainingtheinformationforasinglepolypeptideormolecule,includingtranscribedbutnon-codingAgenetic unitdefinedbya cis-transtest.Forallpracticalpur e,itissynonymous withthe ChemicalnatureofRNA (Ribonucleic Acid)Polynucleotides多聚核苷酸contains:Andbasese +phosphoric acid+ Twopurinesadenin

5、eandguanine腺嘌呤和鸟嘌呤ChemicalnatureofRNA (Ribonucleic Acid)Polynucleotides多聚核苷酸contains:Andbasese +phosphoric acid+ Twopurinesadenineandguanine腺嘌呤和鸟嘌呤 : , , StructureofThe two strands of DNA molecule are double helix of antiparallel strands, held together by Hydrogen bond formation (Guanine Cytosine,Ad

6、enine Thymine). Bases stackinside the helix to excludes water and stabilize themselves. One BASEPAIR =0. 34 nm, complete turn of helix 3.4nm.AndeachturncontainsaMajorandFor single strand of DNA, there exists direction or polarity 5to 3. And the backbone ofchargedphosphateresidues.Sugarsare linkedbya

7、 phosphodiesterbond,and5methylgroupis linked to the 3 OH group.&JonesRNAdiffersfrom1.RNAhasa sugare,whenDNAhasa sugar RNAcontainsuracil(U),whenDNAhasthymineRNAmoleculeissingle-stranded,whenDNAisdouble-4.RNAmoleculeismuchn 5.escontaingenesmadeofRNAinsteadofGenome is the complement of genetic informat

8、ion unique to each species anism, equivalenttotheDNAofahaploidsetofchromosomestDNAdenaturation(melting):thedoubledstrandsofDNAyseparatedeachOnce the two DNAstrands have separated, the t result from are grey decreased, which changes the electronic nature of the bases and increases their UV absorbance

9、 (260nm).Tm isthetemperatureatwhichtheshiftinabsorbanceishalfcompleted.MoreGC,higherGenomesfromdifferentSpecieshavedifferentGCDNArenaturation: when we slowly cool a denatured DNAsolution, DNAregained the ofthedoubletionKinetics复性动力学tion: DNA is o of a dred bp, heated to o strands,thenallowedtorenatu

10、reduringCot1/2:the valuewhen50%renaturation has occurredwhichcanbeused toestimate tionKinetics复性动力学tion: DNA is o of a dred bp, heated to o strands,thenallowedtorenatureduringCot1/2:the valuewhen50%renaturation has occurredwhichcanbeused toestimate the length uniqueDNAinaCo:theoriginalconcentrationo

11、fdenaturedAhighconcentrationofDNAincubatedforashortertime=alowconcentrationofDNAincubated for a longer timeHigherCot1/2valuesindicategreatergenomeCvalueSizeofhaploidgenome=C-C-value um C-values t some less anisms may be nmoreC-valueparadoxisexplainedbyrepetitive.Non-nuclearThemitochondria线粒体andchlor

12、oplasts(叶绿体alsohaveaDNAgenomeorThese resemble procaryotic genomes (likely due butaremuchosymbiotic origin of MethodsofMolecularMolecularcloningClones:a groupofidenticalcellsGenecloning:ProducemanyidenticalcopiesofaUsesofgeneclones:Togetenoughamountofa geneforGOI:Geneerest)PrincipleofgeneIn Vitro (生物

13、体外) : PCR allows sequencing or use of the lification of DNA to enable lified DNA in specific protocols In Vivo (生物体内) : Produce ties of these genes in bacteria by linking genes to small bacterial or phage DNAs and inserting obacterialhosts,makesuretheforeigngenecanPCRPolymeraseChaineofaPCR:tomakeahu

14、genumberofcopiesofaThere are 3 major steps in a PCR,PCRPolymeraseChaineofaPCR:tomakeahugenumberofcopiesofaThere are 3 major steps in a PCR, which are repeated for 30 or 40 automatedcycler,whichcanheatandcoolthetubeswiththereactionmixturet is done on yshortDenaturationat94oC:doublestrandopenstosingle

15、strandedDNA Annealing at 50-60oC: primers bind to the DNAtemplateat 72oC: The bases (complementary to the template) are coupled to the primer on the 3 side (the polymerase adds dNTPs from 5 to 3, reading the template from 3 to 5 basesareaddedcomplementarytotheRequirementsfora Sequenceinformationofth

16、eGOIfordesigningprimers Appropriate template (DNA)Mg2+Buffer(pHandsaltdNTP(materialsformakenewDNARestrictionEndonucleases TheenzymespreventofforeignDNA(restrictviralCharacteristicsofRestrictionEndonucleasesREs recognize specific nucleotide and cleave both strands of the DNAthose.Recognition for many

17、 enzymes are the same on both strands. Such aresaidtobepalindromic(回文序列Commonly used REs always cleave the DNA strands at a fixed recognition sequence.Productsofcleavage:flushendsor5or3ition relative to Isoschizomers (同裂酶): enzymes originated from anism and recognize sequence. They could cut the seq

18、uence at same or different sites, Isocaudomers(同尾酶some REproducecompatibaleoverhamgs,eventhoughtheirarenotRestriction-modificationsystem(R-MTo avoiddestroying thehostcells own DNA,restrictionendonucleasesare paired with methylase t recognize and methylate the same DNA sites.Thetwoenzymes,therestrict

19、ionendonucleaseandthemethylases,arecalledanR-MainingrestrictionafterDNAdam:adeninemethylase,methylatestheN6 dcm:cytosinemethylase,methylatestheitionoftheadeninieitionofernalcytosinehedinucleitiderecognitionsequence. he trinucleitide.CpG:methylatestheCpNpG:methylatesthedam:adeninemethylase,methylates

20、theN6 dcm:cytosinemethylase,methylatestheitionoftheadeninieitionofernalcytosinehedinucleitiderecognitionsequence. he trinucleitide.CpG:methylatestheCpNpG:methylatestheitionofthecytosineitionofthecytosineMethylation-sensitiveRE:REcannotcutifcorrespondingmethylationhappened Host Stains: labeled its me

21、thylation characteristicsDpnI:cutonlymethylatedbinantbinant DNA is s been created lly. DNA from two or more binantoa StepsofmakingCut,ligate,binantoa hostVectors（载体）carrierswhichallowreplicationEssentialcomponentsofa binantORI子thesitewhereDNAreplicationbeginstoensurereplicationofforeignAntibioticmar

22、kergene:toselectthehostcellswiththeMCSMultiplecloningsite,多克隆位点siteallowingtheinsertionofforeignTypesofPlasmidsareextra-chromosomalDNAelements. Phages containing single-stranded DNAthe lchromosomescontainall the ta DNAneedsto functionasa chromosome lBAC:bacteriallPlasmid质粒Plasmids are circular, doub

23、le-stranded DNA molecules some eukaryotic cells.t exist in bacteria he nuclei CharacteristicsofPlasmid:Small+Usuallycarryonlyoneorafewgenes+CircularHaveasingleoriginofreplication,canreplicatePlasmidsarereplicatedbythesametreplicatesthebacterialPlasmidDNAreplicationratePlasmidDNAreplicationratetofthe

24、 tofthe -multiple-singlePlasmidsenterthebacterialcellwithrelativePhages噬菌体Phageisa naturalvectortbacterialDNAfromonecelltoAdvantagestheinfectionstohostcellsareoremodate(提供oreforeignDNA(canbeusedtomakeCosmidscosPhages噬菌体Phageisa naturalvectortbacterialDNAfromonecelltoAdvantagestheinfectionstohostcell

25、sareoremodate(提供oreforeignDNA(canbeusedtomakeCosmidscossite-carryingplasmidCosmids: engineered phages which 载体modate up to 50kb inserts. It contains the cos (cohesiveends)ofphageDNA,whichallowtheDNAtobe ophage CosmidsalsoheORIof aplasmid,andbehave splasmidsandIt does not replicate as phage, but as p

26、lasmid (contains a plasmid ORI). It is only infectious lChromosomesYAC: Avector used to clone DNAfragments (up to 400kb). It is constructed from centromeric,andreplicationoriginneededforreplicationinyeast: omere which is located at each chromosome end, protects the linear DNA degradationbyCEN: The c

27、entromere(着丝粒) whichis the entsite formitotic spindle（纺缍体pullsonecopyofeachduplicatedoeachnewdaughterARS (autonomous replicatingsequence ): specific DNA machinerytoassembleontheDNAandmoveatthereplicationt allow the DNA EssentialcomponentsofYACCentromers omeres ) and autonomous replicating sequence m

28、arkers for identifying cells containing the YAC vector, and recognition sites of DNA is partially digested by EcoRI and the YAC vector is cleaved by EcoRI Ligate the cleaved vector segments with a digested DNA fragment to form an artifi Transformyeastcellstomakealargenumberofl lchromosomesBAC: A vec

29、tor used to clone DNA fragments of 100 to 300 kb insert size in E. coli cells. It basedonthenaturallyctorplasmidfoundinE.Enzymesusedincloning:Restrictionenzyme,Alkalinephosphatase,DNADNAlibrary:acollectionofclonedDNAfragments,includin cDNA: complementary DNA, a DNA copy of RNANAlibraryandgenomiccDNA

30、library: a set of clones DNAlibrary:acollectionofclonedDNAfragments,includin cDNA: complementary DNA, a DNA copy of RNANAlibraryandgenomiccDNAlibrary: a set of clones representingas many sible of the mRNAs in a given cell ata giventime(temporalandspatialReversetranscriptionReversetranscriptase:RNA-d

31、ependentDNApolymerase RT-PCR: Reverse transcriptase PCRMakingacDNAUsingoligo(dT)asaprimer,producestrandofRNase H: it is a kind of enzyme which can partially digest mRNA, yielding a set of RNA base-pairedtostrandDNApolymerase:tobuildthesecondstrandcDNAontheRNATerminaltransferase:addoligo(dC)softhecDN

32、Aanneal(退火tocomplementaryoligo(dG)endsofasuitableRT-PCRtocloneasingleUseareverseprimerwithHindIIIsiteatits5-endto-strandcDNAPCRreactionusingthesynthesizedcDNAastemplate(forwardprimerhasBamHICutthePCRproductswithHindIIIandBamHI,ligateo a RACE: lification of cDNA ends, a method for extending a partial

33、 cDNA to its 5- 3-endPCRcDNA末端快速克隆的技术TomakethewholelengthofcDNA:5-RACEand3-5-HybridizepletecDNAtomRNA,useRTtoextendthecDNAtothe5-endofAddCresidualstothe3-endoftheextendedUseanoligo(dG)primertosynthesizethesecondstrandofcDNA(terminaltransferase) Perform PCR using known 3sequence and oligo(dG) as prim

34、ers3-AsimilarproceduretoextendthecDNA Using an anchor primer (锚定引物)he3-hatcase, there is no needto tail the 3-endof the cDNA with terminaltransferase because mRNA already contains poly(A) and the anchor sequence; thus, the reverse primer would be the anchor primer.MethodsofexpressingclonedUsing cDNA

35、 sequence to make the clones: because the rons has been removed in CouldusingantibodytoidentifyclonesfromtheexpressingvectorshavetwoMethodsofexpressingclonedUsing cDNA sequence to make the clones: because the rons has been removed in Couldusingantibodytoidentifyclonesfromtheexpressingvectorshavetwoe

36、ssentialStrongbacterialpromoter +AomebindingsitenearaninitiatingATG proteins融合蛋白）TheproductscontainextraaminoEukaryoticAdvantagesof“Eukaryoticproteinsmadeineukaryotic(1)Proteinwillbefoldedproperly;(2)proteinsaremodifiedinaeukaryoticUsing a shutter vector tcan replicate in both bacteria and eukaryoti

37、c cells), the initial isusuallydoneinE.Coli,thenbinantDNAistransferredtotheeukaryotic杆OtherEukaryoticTiplasmid:itcantransportforeignoplantcellsandensuretheirreplicationNativeT-DNAplasmid:Crowngall冠狀肿瘤CrownGallDiseasehastwomajorelements:(1) eneswhichsynthesizeextraplant hormones to stimulate neoplast

38、ic growth of infected cells, and (2) opine genes which unusualaminoacidstcanonlybemetabolizedbyMolecularGelelectrophoresis凝胶电泳Ionexchangechromatography （离子交换层析Gelfiltrationchromatography （凝胶过滤层析 (DNAandRNAmoleculesarenegativecharged,theymigratetowardtoitiveTheratesofmoleculesaredifferent.Commonly,sm

39、allermoleculesmoveThe mobilities of fragmentsare correlated with the log ofmolecular weight or the number of PFGE (pulsed-field gel electrophoresis, 脉冲场凝胶电泳) was invented for the separation largesizeDNA,the linear relationship nthe logofa DNAssize anditsmobilitynoexistswhentheDNAmoleculesareverylarg

40、e.LargerDNAmoleculesareveryeasytoThe method uses pulses of current, with relative long he forward direction and he ite,orevensideways,PAGE: polyacrylamide gel electrophoresis, used to separate polypeptides according to their mass (molecular weight).SDS: treat proteins with detergent (SDS) to denatur

41、e te) the subunits of SDS coats all the polypeptides with negative charges, so they all movePAGE: polyacrylamide gel electrophoresis, used to separate polypeptides according to their mass (molecular weight).SDS: treat proteins with detergent (SDS) to denature te) the subunits of SDS coats all the po

42、lypeptides with negative charges, so they all move to the And it masks the natural charges of the subunits, so they all electrophorese according to their molecular masses, not by their native charges.algelelectrophoresis双向凝胶电泳Step 1: the mixture of proteins is electrophoresed through a narrow tube g

43、el containing molecules holytes (双性电解质), which set up a pH gradient from one end of the tube the other. A negative charged molecule will electrophoreses towards the anode until reachesitsisoelectricStep2: The gel separated from step 1 is placed at the top of a slab gel for ordinary SDS-Proteinswillb

44、eresolvedaccordingtotheirIon-exchangechromatographyIEC, 离子交换层析Usearesin树脂)toseparateaccordingtotheirGelfiltrationchromatography凝胶过滤层析SeparatesmoleculesbasedontheirColumnisfilledwithporousresin多孔渗水树脂whichletsinsmaller,butlargerones.AndthesmallermoventhelargerLabeledtracers标记示踪Detection of the ties of

45、 used in molecule biology experiments requirestheuseoflabeledRadioactive tracers can be detected by autoradiography of liquid scillationcounting Nonradiosctive labeled tracers: chemiluminescence (化学发光）Nucleicacidhybridization Principle: a single-stranded nucleic acid can form a double helix with ano

46、ther single strand of complementary base sequence.Southernblot:identifyingspecificDNANorthernblot:identifyingspecificRNAProcedureofSouthernGenomicDNAdigestionwithrestrictionGelelectrophoresisofthedigestedDNADNAfragmentsaredenaturedandoanitro-UsecertainlabeledprobestohybridizetheWashouttheextraprobe,

47、autoradiographyorothermethodstodetectthelabeledliteDNA&DNArlite DNA:Aportionof DNAin eukaryoteswhose density differstof the majority DNA,tconsistsofshort,liteDNA&DNArlite DNA:Aportionof DNAin eukaryoteswhose density differstof the majority DNA,tconsistsofshort,repeatingHighly repetitive, very andem

48、arrays, concentrated near the centromeres and forms large part of heterochromatin, as separate band in buoyant density gradient, no function exceptsibleroleinkinetochoreDNA ing: the use of highly variable regions of DNA to identify particular usinga liteDNAasa lite DNA(DNA): DNAwhich are highly poly

49、morphic, 5-100 bp upto3000 DNA):1-5bpsequencerepeatedto50-100bpliteDNA(SSR(Simplesequencerepeat)=STR(ShortTandemliterepeatsarethebasisoftheDNAing印迹) StepsofDNACutDNAwithrestrictionGelelectrophoresed,denaturedandThe blot was hybridized wilabeled minisaliteDNAprobe Detection of the labeled bandsDNARFL

50、P,restrictionfragmentlengthDNA:usingaspecificprobetoDNARFLP,restrictionfragmentlengthDNA:usingaspecificprobetoidentifythedifferenceamongindividualsinoneNorthernA Northern blot is similar to a Southern blot, but it contains electrophretically separation RNAs instead of DNAs.Itisusedtostudytheofagene,

51、andneedaloadingThe nggene isusedasloadingInsitu-hybridizationISH, 原位杂交ISHisusedtolocategeneinFISH:fluorescenceinsitu-hybridization荧光原位杂交HybridizelabeledprobestowholechromosomestolocatespecificDNAInsituRNAhybridization:Hybridizelabeledprobestoparticularestosee of DNATheSangererminationsequencingUse d

52、ideoxynucleotides (双脱氧核苷酸）to terminate DNA synthesis, yielding a series of fragmentwhosesizescanbe measuredbyRestrictionRestriction : Cut the DNA in question with two or more restriction enzymes in RestrictionRestriction : Cut the DNA in question with two or more restriction enzymes in reactions, me

53、asure the sizes of the resulting fragments. It is used determininginwhichiontheinsertionwasothe (PCR-basedsite-directedWhyS1 ?Locatethe5-or3-endsofRNAs(startingands of fytheamountofa givenRNAincellsatagivenHowdoesitcarriedLabelasingle-strandedDNAtcanhybridizeonlytothetranscriptHybridizationoftheprob

54、e(single-strandedDNA)totheDigestionbyS1nuclease(specificallydegradesingle-strandedpolynucleotides) The transcripts hybridized with probe was protected from digestionNuclearRun-ontranscription转录活性测定IsolatenucleifromcellsandallowthemtoextendinNuclearRun-ontranscription转录活性测定Isolatenucleifromcellsandal

55、lowthemtoextendinvitrothetranscriptswhichhadalreadyin vivo. In a isolated nuclei, RNA t already initiated transcription in vivo “run-on”,whiletheinitiationofnewRNAchainsdonotThisisusedtomeasuretranscriptionrates,orfindwhichgenesaretranscribedinReportergeneAreportgenesproductisveryeasilytoReporterLac

56、Z(-Cathenicol acetyl transferase 氯霉素乙酰转移酶OtherreportergusA(uidA)(-glucuronidase, 葡萄糖甘酶); EMSAElectromobilityShiftAssays) FeaturesofDNA ( ()DNApolymerasesaretemplate-dependentandprimer-AllDNApolymerasessynthesizehe5to3direction,readingthetemplate3to PrimerdependenceDNApolymerasecannotinitiateDNAsynth

57、esiswithouta10-12ntlongRNAWaysofDNABidirectionalreplication:TworeplicatingforksmoveinitedirectionsawayfromtheUnidirectionalreplication:OnlyWaysofDNABidirectionalreplication:TworeplicatingforksmoveinitedirectionsawayfromtheUnidirectionalreplication:OnlyonereplicatingforkmovesfromtheRolling circle rep

58、lication: one strand of a double-stranded circular DAN asthetemplateforelongationoftheotherstrandataact & ORI:AuniqueDNAsequenceatwhichDNAreplicationis子alltheDNAreplicatedfromoneoriginofProkaryotic chromosomes are circular. DNA replication begins at a single origin po and proceeds around the circle

59、in both directions.In eukaryotic cells, the chromosomes are linear. They are also much larger. In order to keep replication within a reasonable time frame, these large chromosomes begin replication at several origin po s. Each replication origin proceeds bi-directionally, again with leading and stra

60、ndsaccordingtothesameUnidirectionalE. Colis plasmid colE1 contains a EcoRI site, linerized plasmid DNA is used as template, it be tthereplicationtakesplace inRollingCircleCircularDNAcanreplicatebyarollingcirclemechanism:onestrandofadouble-strandedDNAnickedand3-endisextended,usingactDNAastemplate.Whe