基因工程原理Chapter 2

Chapter2

Genemanipulation:anall-embracingtechnique2-1Introduction1.Somedevelopmentsenablesciencetoleadforwardandincreasethepotentialforinnovation2.GenemanipulationSophisticatedinvivogeneticsAppliedto

SophisticatedinvitrotechniquesNewcombinationsofheritablematerialareconstructedbytheinsertionofnucleicacidmolecules,thoughvirus,bacterialplasmidorothervectorsystemoutsidecell,thentheincorporatedvectorentershostorganismandcanmultiplyinsidethehost,showingunnatural.

Simplyspeaking,genemanipulationpermitsstretchesofDNAtobeisolated(cloned)fromtheirhostorganismandpropagatedinthesameoradifferenthost.

2-2Sequencing

Steps:cloningsequencingscanningdeducingproteinsequencebasedonnucleotidesequence.ScanningIfafragmentsequenced,thenputthesequenceintoasearchingengineontheinternet:BLAST(/),forsearchingitshomologueandpossiblefunction.BLAST(BasicLocalAlignmentSearchTool,局域相似性比對分析工具)1.Nucleotide-nucleotideBLAST,blastn);2.Nucleotidequery-protein,blastx

Butremember:FASTAformatisrequired.HowtoBLAST?ResultforthesearchGeneprediction1.ORFfinderisusuallyusedforthispurposeatthebeginning;2.Functiondomainprediction.3.Manybio-softwareareconvenientonlineoravailablefromsomecompanies:Bio-edit,DNAman,etc.Ifyouhaveaspecificfragmentorgene,youwillknowitsdigestionsite,startandstopcodons,invertedpalindromes,sequencerepeats,Z-DNAandsoon.Promoterprediction/isavailableontheinternet;The2kbsequencebeforetheinitiationcodonofanORFshouldbeusedfortheprediction.DeducingproteinsequenceBysoftware!Itissoeasyjob!Whatyoudoisjustto“Click”somebuttonsinyourcomputer.Motif:continuoussequenceofaminoacidsorDNAsoastoformafragment.Itisverydifficulttopredictaproteinfromthreedimensionalstructurefromaamotifs.However,comparemorecrystallographicdataofproteinscompareaasequenceofproteinusecomputersummarize:ahighdegreeofhomologysuggestssimilarityinfunction.2-3.Invivobiochemistry

2-4.Thenewmedicine2-5.Biotechnology:thenewindustry2-6.ThecentralroleofE.coliAGeneticOde遺傳賦

(orAMelanColiTale,一個不起眼的大腸桿菌的述說)Iusedtobeacoli,aswildaswildcouldbe.TheycalledmePhotoTrophic,whateverthatwouldbe.TheykeptmepureandsimpleandcompletelyfreefromfaultsAndfedmeonthesimplestfood…glucoseandcommonsalts.ThenLederbergandTatumcameandputmeinthesunAndwatchedmeverycloselytoseewhatharmthey’ddone.Althoughtheyhadn’tkilledme,theyhadreallyhurtmyprideAndthoughIlookedquitenormalIwasquiteupsetinside.NextdaytheytriedtofeedmewithmynormalsortoffoodButtheyfoundIcouldn’tuseitinthewayIalwayscould.GlucoseIcouldmetabolize-inthatIwasproficient.ButinsynthesizingvalinetheysoonfoundIwasdeficient.Theycouldn’tfindtheirvalinesotheywenttobiotinAndtilltheythoughtjustwhattodotheykeptmedietin’.ThenforesightanddiscernmentmadethislecturerandProf.EnrichmyfoodwithOxacubeandcallmeOxoTroph.Theycalledanotherdoctorandtheyalldiscussedmycase.AnddecidedthatmyDNAmusthavedisplacedabase.TheyallseemedquiteexcitedandIheardDocTatumsay,AnotherdoseofsunshinemightupsetmoreDNA.Theygaveme80secondsofthebr