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Lecture 7

55-213 Lecture 7 notes.pdf

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Department
Biological Sciences
Course Code
55-213
Professor
Hubberstey

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Lecture 7 20130205 348 AM Importantconceptsforweek4 GeneticengineeringandrelatedtechnologiesDNASequencingmicroarrayswesternblots HowdoesIlluminasequencingwork RevolutionaryDNAnanotechnologieso LastclasswetalkedaboutgeneticengineeringNowwelltalkaboutthetechnologiesinvolvedinitandrelatedtechnologiesthathaveapplicationstomolecularbiologyprimarilyDNAsequencingbutalsointhefieldofmicroarraysandWesternblotso ThesimplemicroarraytechnologyhasrevolutionizedmolecularbiologyManypeopleknowaboutittodaybutthiswasntthecase5yearsagoo DNAsequencinganothernewtechnologyo MostofthislectureisntcoveredinthebookThisisbecausewhenitcomestothiskindoftechnologynotextbookcanbewrittenfastenoughandnotbealreadyoutofdatewhenpublishedo TodaywewillcoverthemostuptodatetechnologiesrelatingtoDNAsequencingaswellaswhereitsgoingtogointhenextfewyearsDNAnanotechnologiesQ9o Definitionofnanotechnologyissomethingverytiny10mItsrevolutionizingcomputersandmanyfieldotherthanmolecularbiologyinregardstoDNAsequencingGFPDiscovery2008NobelPrize NobelPrizeinchemistry2008DoctorsShimomouraTsienandChalfiereceiveditforthediscoveryanddevelopmentofthegreenfluorescentproteinGFP Researchersincellbiologyandmolecularbiologyusedtoreplyonradioactive3235125isotopesabout15Q20yearsagoSomeexamplesarePSIandafewothersusedtomonitorwhereDNAorproteinisinacellItworksreallywellinemittingradioisotopesthatyoucanvisualizeusingXQrayfilmoranothertechnologybutithasmajordrawbacksyouhavetohavespeciallicensestouseityoumustdisposeofitverycarefullyasitsradioactivewasteitsasafetyissueitsahazardanditsarealpainforsupervisorsinthelabWhydoesnttheprofessoruseradioisotopesanymoreinhislab1reasonthankstothecreationoffluorescentproteins WherewerefluorescentproteinsfirstisolatedJellyfish ManymarineorganismsactuallycontaingenesthatproducethisautofluorescentcompoundIfyoureonadarkbeachyoucansometimesseethisfluorescenceglisteningfromthewater ThediscoveryofgreenfluorescentproteinswasawardedaNobelPrizein2008 ThereprobablyisnomolecularbiologylabtodaythatdoesntuseGFP GFPfluorescesonitsown MolecularbiologyhasnowtakenthisGFPandgeneticallyengineeredmutationsinitthatallowittofluorescenceorangeyellowredandalmostanycolorinthelightspectrumThusnowtheresRFPYFPetcTheyarentasnaturalasGFPtheyresynthesizedinthelab ThewonderfulthingisthatyoucanseetheseinthelabwiththenakedeyeyoudontneedanyXQraytechnologyoranythinglikethat TheothergreatthingisthatwhencellsareexpressingthisyoucanlookattheminrealtimeYoucanseeithappeningiemovingunderthemicroscopewithoutkillingthecellPreviouslyyouhadtokillthecellandfixitinordertoseewhatsgoingoninit YoucanmakeGFPfusionswithotherproteinsveryusefulinlab AnotherthingyoucandowithGFPismakeanimalstransgenicpetsNobel Prize in chemistry 2008 Drs Shimomoura Tsien and Chalfie for the discovery and development of the green fluorescent protein GFP TransgenicPets ZebrafishexpressingdifferentfluorescentproteinsGFPYFPRFP wwwglofishcombuyaspx TakeazebrafishDNAandmicroinjectacopyofGFPinitThiscopygetsmicrointegratedintothefishschromosomeandgettransgenicfishproducingGFPYFPRFPetc Youcanbuythesefish ThesefishscolorsarecompletelyartificialtheyregeneticallychangedineverysinglecellwhichnowexpressesGFPataveryhighrateInsomecells20ofproteinbeingmadeisGFPTherearenodevelopmentalissueswiththesefishthankfully Oneexampleofbiotechnologicalapplicationofthistechnologyo Transgenic PetsZebrafish expressingDifferent fluorescentProteins GFP YFP RFPhttpwwwglofishcombuyaspTerminology ThebiggestissueofgeneticengineeringisthatyoucanmanipulateDNAandputitintojustaboutanyorganismaswithtransgenicpetsThereareactuallycowsouttherethatarecompletelygreenthankstoGFPexpressionwithouthavinganysignificanthealtheffects YouneedsomethingtomovetheDNAaroundInthe1980scloningvectorswerecoined CloningvectorcirculardsDNAplasmidthatisusedasavehicletotransferDNAintoorganisms
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