《基因的分子生物学》3-基因文库和筛选（英语教学课件）非AI生成.ppt

SectionI

Genelibrariesandscreening

LibrariesofDNAMoleculesCanBeCreatedbyCloningADNAlibrary(文库)isapopulationofidenticalvectorsthateachcontainsadifferentDNAinsert.Genomiclibraries(基因组文库)derivedfromtotalgenomicDNAcleavedwitharestrictionenzyme.

返回Figure20-8ConstructionofaDNAlibrary

I1GenomiclibrariesRepresentativegenelibrariesSizeoflibraryGenomicDNAKeynotes:BACK

Genelibrary:acollectionofdifferentDNAsequencefromanorganismeachofwhichhasbeenclonedintoavectorforeaseofpurification,storageandanalysis.GenomiclibrariescDNAlibrariesGenelibrary(madefromgenomicDNA)(madefromcDNA-copyofmRNA)

Makingarepresentativelibrary---ContainingalltheoriginalsequencesCertainsequenceshavenotbeencloned. Example:repetitivesequenceslackingrestrictionsites2. LibrarydoesnotcontainsufficientclonesMissingoriginalsequenceToolongforthevectorusedBack

Sizeoflibrary(ensureenoughclones)mustcontainacertainnumberofrecombinantsfortheretobeahighprobabilityofitcontaininganyparticularsequenceTheformulatocalculatethenumberofrecombinants:N=ln(1-P)ln(1-f)P:desiredprobabilityf:thefractionofthegenomeinoneinsert

Forexample:foraprobabilityof0.99withinsertsizesof20kbthesevaluesfortheE.coli(4.6×106bp)andhuman(3×109bp)genomesare:NE.coli==1.1×103ln(1-0.99)ln[1-(2×104/4.6×106)]Nhuman==6.9×105ln(1-0.99)ln[1-(2×104/3×109)]Easytomakegoodgenomiclibrariesfromprokaryotesinplasmidswheretheinsertsizeis5-10kb,asonlyafewthousandrecombinantswillbeneeded.Back

GenomicDNAlibrariesPurifygenomicDNAFragmentationofDNA:physicalshearingandrestrictionenzymedigestioneukaryotesprokaryotesClonethefragmentsintovectorsBack

Tomakearepresentativegenomiclibrar