目的基因的获得 PPT课件.pptVIP

目的基因的获得

ObtainingthetargetgeneFromthegenomicDNAFromthegenomicorcDNAlibraryFromthePCRproductsFromartificialsynthesis目的基因的获得

1.AlkalineisolationofDNALysozymeGlucoseEditicacid,EDTANaOHSodiumdodecylsulfate,SDS3MNaAc(pH4.8)EthanolorisopropanolNaAcorNaClRNase,SDS,KAcPhenol/chloroform/isoamylalcohol目的基因的获得

PurificationofplasmidDNAusing

DNAisolationkit目的基因的获得

2.ConstructionofgenomiclibraryCharacteristicsofagoodgenomiclibrary:a.representthewholegenomeb.stable(2)Factorsshouldbeconsidered:a.thenumberofclonesb.thetypeofvectortobeused目的基因的获得

GenomiclibraryN=ln(1-P)/ln(1-a/b)NisthenumberofclonesrequiredPisthedesiredprobabilityofaparticularsequencebeingrepresentedaistheaveragesizeoftheDNAfragmentstobeclonedbisthesizeofthegenome(expressedinthesameunitsasa)目的基因的获得

Sizeandstructureofsomehumangenes目的基因的获得GeneGenesize（kb）Numberofexons%exonsInsulin1.4333β-globin1.6338Serumalbumin181412BloodclottingfactorVIII186263CFTR(cysticfibrosis)230272.4Dystrophin(musculardystrophy)2400790.6

GenomicSizeinsomeorganisms目的基因的获得OganismGenomesize(Mb)Escherichiacoli(bacterium)4.6Saccharomycescerevisae(yeast)12.1Drosophilamelanogster(fruitfly)150Homosapiens(man)3000Musmusculus(mouse)3300Nicotianatabacum(tobacco)4500Triticumaestivum(Wheat)17000

Genomiclibrarysizesforvariousorganisms目的基因的获得OrganismGenomesize(kb)No.clonesN,P=0.9520kbinserts45kbinsertsEscherichiacoli4.0X1036.0X1022.7X102Saccharomycescerevisae1.4X1042.1X1039.3X102Arabidopsisthaliana7.0X1041.1X1044.7X103Drosophilamelanogster1.7X1052.5X1041.1X104Stronglyocentrotuspurpuratus8.6X1051.3X1055.7X104Homosapiens3.0X1064.5X1052.0X105Triticumaestivum1.7X1072.5X1061.1X106

PartialdigestionandfractionationofGenomicDNA目的基因的获得

CloningSau3AfragmentsintoaBamHIsite目的基因的获得(a)BamHI5’-GGATCC-3’3’-CCTAG