目的基因的獲得課件

目的基因的獲得ObtainingthetargetgeneFromthegenomicDNAFromthegenomicorcDNAlibraryFromthePCRproductsFromartificialsynthesis目的基因的獲得1.AlkalineisolationofDNALysozymeGlucoseEditicacid,EDTANaOHSodiumdodecylsulfate,SDS3MNaAc(pH4.8)EthanolorisopropanolNaAcorNaClRNase,SDS,KAcPhenol/chloroform/isoamylalcohol目的基因的獲得PurificationofplasmidDNAusing

DNAisolationkit目的基因的獲得2.ConstructionofgenomiclibraryCharacteristicsofagoodgenomiclibrary:a.representthewholegenomeb.stable(2)Factorsshouldbeconsidered:a.thenumberofclonesb.thetypeofvectortobeused

目的基因的獲得GenomiclibraryN=ln(1-P)/ln(1-a/b)NisthenumberofclonesrequiredPisthedesiredprobabilityofaparticularsequencebeingrepresentedaistheaveragesizeoftheDNAfragmentstobeclonedbisthesizeofthegenome(expressedinthesameunitsasa)目的基因的獲得Sizeandstructureofsomehumangenes目的基因的獲得GeneGenesize（kb）Numberofexons%exonsInsulin1.4333β-globin1.6338Serumalbumin181412BloodclottingfactorVIII186263CFTR(cysticfibrosis)230272.4Dystrophin(musculardystrophy)2400790.6GenomicSizeinsomeorganisms目的基因的獲得OganismGenomesize(Mb)Escherichiacoli(bacterium)4.6Saccharomycescerevisae(yeast)12.1Drosophilamelanogster(fruitfly)150Homosapiens(man)3000Musmusculus(mouse)3300Nicotianatabacum(tobacco)4500Triticumaestivum(Wheat)17000Genomiclibrarysizesforvariousorganisms目的基因的獲得OrganismGenomesize(kb)No.clonesN,P=0.9520kbinserts45kbinsertsEscherichiacoli4.0X1036.0X1022.7X102Saccharomycescerevisae1.4X1042.1X1039.3X102Arabidopsisthaliana7.0X1041.1X1044.7X103Drosophilamelanogster1.7X1052.5X1041.1X104Stronglyocentrotuspurpuratus8.6X1051.3X1055.7X104Homosapiens3.0X1064.5X1052.0X105Triticumaestivum1.7X1072.5X1061.1X106PartialdigestionandfractionationofGenomicDNA目的基因的獲得CloningSau3AfragmentsintoaBamHIsite目的基因的獲得(a)BamHI5’-GGATCC-3’

3’-CCTAGG-5’Sau3AI5’-NGATCN-3’

3’-NCTAGN-5’(b)BamHIGATCCCCTAGGG(c)Sau3AICTAGCTAGGATCGATC(d)CTAGGGGATCCCTAGGATCCAnnealVectorInsertVectorPreparinggenomiclibrariesinbacteriophageλvector

目的基因的獲得CloningofhumanDNAusingplasmidvectors目的基因的獲得CloningoflargesegmentsofgenomicDNAusingcosmids目的基因的獲得PreparationofmRNAby

affinity

chromatographyusingoligo(dT)-cellulose目的基因的獲得Isolationofpoly(A)RNA目的基因的獲得mRNAabundanceclasses目的基因的獲得SourceNumberofdifferentmRNAsAbundance(molecules/cells)Mouselivercytoplasmicpoly(A)+RNA9120007003001150015Chickoviductpolysomalpoly(A)+RNA110000074000125005Synthesis

ofcDNA目的基因的獲得Oligo(dG)-primedsecond-strand

cDNAsynthesis目的基因的獲得OnemethodofconvertingmRNAtodouble-strandedcDNA目的基因的獲得Cloningofdouble-strandedcDNAinphagevectors目的基因的獲得Screeningalibrarywithanucleicacidprobetofindaclone目的基因的獲得3.PCR------DegeneratePrimerPCR

SelectingthesequenceaminoacidsequencePhe-Leu-Pro-Ser-Ala-Lys-Trp-Ala-Tyr-Asp-Pro

numberofcodons26

4

64214224peraminoacidavoidbettersequence(b)MixedprobesynthesisAlaLysTrpAlaTyrAspProGCAAAATGGGCATACGACCCGGGTTCCTTNumberofpossibilities4x2x1x4x2x2x1=128

Usinginosineasthedegeneratebase

AlaLysTrpAlaTyrAspProGCIAAATGGGCITACGACCCGTT

Numberofpossibilities1x2x1x1x2x2x1=8目的基因的獲得DirectlycloningofPCRproducts目的基因的獲得5’5’HindIIIEcoRIHind