Chapter20Biotechnology

I. RecombinantDNA
A. ThecreationofnewDNAsegmentsthatarenotfoundtogetherinnature
B. Theisolationandmanipulationofgenesallowsformoreprecisemoreprecise
geneticanalysisaswellaspracticalapplicationsinmedicine,agriculture,and
industry
II. MakingrecombinantDNA
A. IsolatetheDNA
1. cutitwithrestrictionenzymes
2. ligateitintoacloningvector
3. transformedDNArecombinantmoleculeintohostcell
4. eachtransformedcellwilldividemanytimestoformacolonyofmillionsof
cells,eachofwhichcarriestherecombinantDNAmoleculeorDNAclone
III. CuttingtheDNA
A. DNAcantbecutmechanically
B. userestrictionenzymeswhicharescissorsofmoleculargenetics
C. Restrictionenzymesareethatwillrecognizespecificnucleotidesequences
1. theybreaktheDNAchainatthesepoints
D. avarietyofrestrictionenzymesavailablecommercially
E. mostcutatspecificpalindromicsitesintheDNA
1. sequencethatisthesameonbothantiparallelDNAstrands
2. thesecutscanbestaggeredwhichmakestickyoroverhangingendsora
bluntwhichmakeflatends
IV. JoiningtheDNA
A. onceyouhaveisolatedandatthedonorDNAandthevectorDNAtheyhavetobe
joinedtogether
B. theDNAfrombotharemixedinatube
C. ifbothhavebeencutwithsameREtheendswillmatchupbecausetheyare
sticky
D. useDNAligasewhichistheglueofmoleculargeneticstoholdtheendsofthe
DNAtogether
E. DNAligasecreatesaphosphodiesterbondbetweenthetwoDNAend
V. Restrictionenzymes
A. recognizedsequencelengthvariesbutmostareeither4basecuttersor6base
cutters
B. thesequenceononestrandmatchesthatofthecomplementarystrandreadin
theoppositedirection
C. restrictionenzymesmostlycomefrombacteriaandtheirnaturalpurposeisto
destroyDNAfrominvadingviruses
VI. cloningofrecombinantDNAusingvectors
A. cloningistheprocessofmakingmanyidenticalcellsfromthecellscontianingthe
recombinantDNA
B. thegenepieceintroducedintherecombinantDNAissaidtobetheDNAthatis
cloned
C. recombinantDNAisintroducedintocellsbyavector
D. avectorisameansofdeliveringrecombinantDNAtoanorganism
E. vectorsmusthaveawayofgettingintothehostorganism[transformation]
F. vectorsmusthavesomewayofbeingpropagated
1. sometypesofvectorsremainfreeincellbutarecopiedanddistributedin
celldivision
2. sometypesofvectorshavetheinsertedDNAintegrateallorinpartwith
thehostDNA
G. thevectorDNAsequencemustbeknownenoughsothatrestrictionsitescanbe
accuratelypredictedandused
H. mostcommonly,vectorsareplasmids,viruses,oryeastartificialchromosomes
[YACs]
VII. Plasmidsasvectors
A. mostcommonlyused
B. small,circularDNAmoleculeswithatleastonereplicationorigin
C. mostbacterialcellscontainseveralplasmids
D. someeukaryoticcellshaveplasmidssuchasyeast
E. plasmidsvaryinwhatorganismcanmaintainthem
F. mostplasmidscontaingenesthatareexpressedwithvariationsdependingon
hostcell
VIII. virusesasvectors
A. virusesinfectcellswiththeirDNAsorecombinantDNAinaviruscanbethus
transferredtocells
IX. Yeastartificialchromosomesasvectors
A. eukaryotescansupportandmaintainlargerpiecesofDNAaschromosomes
B. YAGhavetherequiredelementsofchromosomes[centromere,telomeres]and
canbeusedasvectorsforlargesegmentsofclonedDNA
X. CloningSites
A. vectorstypicallyincludeaselectablemarkerandacloningsite
B. selectablemarkersareusuallyageneforaproductthatthehostcellcantmake
uschasanantibioticresistancefactor
C. thesloningsiteonavectorisengineeredwithmanypossiblesitesforREcutting
whereforeignDNAcanbeinserted
D. thepieceofforeignDNAinsertedatacloningsiteissaidtobeclonedandthe
combinedforeignDNAplusthevectoriscalledrecombinantDNA
XI. Screening
A. often,manyclonesaremadewithvariousDNApiecesinserted
B. ScreeningisusedtofindtheDNAofinterest
1. aselectablemarkerisusedtoinsurethatthevectorispresent
2. asecondselectedmarkerisusedtomakesurethatthevectorcontains
theinsertedDNA
3. cellsfromcoloniesthatpassthescreenstothispointareusedas
sourcestomakelargenumberofcells
4. DNAfromthesecellsissubjectedtoothertreatmentstohelpidentifycell
linescontainingtheDNAofinterest
XII. DNAlibraries
A. thefirststepinworkingwiththeDNAofaspeciesistobreakthewholegenome
downintomanageablebits
B. thisisdonebycreatingDNAlibraries
C. vectorsserveasthebooksinaDNAlibrary
1. eachbookhasadifferentpieceofinsertedDNA
D. twomaintypesoflibrariesaregenomiclibrariesandcDNAlibraries
XIII. genomiclibraries
A. rawgenomicDNAisbrokenintofragments
1. sometimesdonemechanically
2. sometimesdonewithRE
3. oftenacombination
B. thebrokenDNApiecesareputintovectorsandthenthevectorsareputintohost
cells
C. thecelllinesaremaintainedforeachlibrarypiece
D. thecelllinesaregivenuniqueidentifiersandDNAprobingcanbeusedto
determinewhatlinescarryparticularclonedsequences
XIV. complementaryDNAlibraries(cDNA)
A. morerefinedapproachthangenomic
B. basedoncodingregionsofDNA
C. mRNAsareisolatedfromacellandconvertedintocomplementaryDNAusing
reversetranscriptase
D. thecDNAisinsertedintovectorsandthelibraryismadeandmortaredsameas
genomiclibrary
E. DNAprobesareusedtofindlinesofDNAofinterest
XV. TechniquesusedtomanipulateandstudyDNAbeforeandaftercloning
A. DNAsequenceamplification
1. smallamountofDNAismadeintoalargeramounttostudy
2. useaPCRpolymerasechainreaction
3. DNApolymerasecanbuildaDNAstrandprovidedthereisatemplate
strand,aprimer,anddNTPsdeoxyribonucleotidesacidsofeachtype:
dATP,ATP,dGTP,dTTP
4. basicsteps:
a) denaturation
(1) heatingaDNAmoleculewillseparatethedoublestrands
intoseparatesinglestrands,breakingthehydrogenbonds
betweenATandGCbasepairstoprovidetemplate
strands
b) annealingofprimers
(1) whentheDNAcoolsbasepairswillreform
(2) ifsmall,specificDNAprimersareaddedinexcess
comparedtoamountoftemplateDNA,theprimerswillbind
tothetemplateDNAstrandskeeptheoriginaldouble
helicesfromforming
(3) Primerextension
(a) DNApolymerasescanaddthedNTPstomakea
complementaryDNAstrandstartingatthe3end
(b) ifthisprocessisrepeatedthroughseveralcycles,
youwillexponentiallymakenewDNAstrands
5. PCRworkswellonlywhentheDNApolymerasecanwithstandthehigh
temps.UsedtoseparatetheDNA
a) THeseenzymeswerefoundinbacterialikeorganismsthat
normallyliveinhotenvironmentssuchashotsprings
b) theseareknownasheatstableDNApolymerases
6. specificprocedureforPCR
a) DNAmeltingisdoneat94Cforaminuteorless
b) DNAannealingisdoneat50Cforaminuteorlessitcanvary
dependingontheDNAprimersused
c) DNAsynthesis[primerextension]isdoneattheoptimal
temperaturefortheheatstableDNApolymerasewhichisusually
around72C
d) theprocessthengoesbacktomeltingandCYcleisrepeatedfor
upto35cycle
7. After4cyclesyouwouldhave2
4
=16DNAmolecules
8. after20cyclesyouwouldhave2
20
=1,048,576DNAmolecules
9. PCRcanbeusedtomakeenoughrawmaterialforcloning,DNAlibraries,
andDNAsequencing
XVI. DNAGelelectrophoresis
A. overallDNAmoleculesarenegativelycharedandwillmigratethroughaviscous
materialsuchasagelifavoltagedifferenceissupplied
B. speedofmigrationisdeterminedbythesizeoftheprotien.
C. thisalsocanbeusedforRNAbutpreventingdegradatinofRNAisachallenge
XVII. Probing
A. DNAandRNAfragmentscanbetransferredtoafilter,denatured,andincubated
withprobesmoleculesthatwillhybridizewithspecificsequences.
B. theprobemoleculesareusuallyDNAfragmentsandaremadeofsome
nucleotidesthatareeitherradioactiveorfluorescentwhichthuslabelstheprobe
1. whenprobeisuseditlabelsthesitesonthefilterwheretheprobeisable
tohybridize
C. ifDNAisonthefilterandbeingprobeditiscalledaSouthernBlotorDNAgelblot
D. ifRNAisbeingprobeditiscalledaNorthernBlotorRNAgelBlot
E. ifproteinsarebeingprobeditiscalledawesternblot[antibodiesand
trippohoophie.
XVIII. DNAsequencing
A. determinedusingspecialnucleotidesandmigrationdifferencesDNAstrands
throughagelbasedonsize
B. specialddNTPsareusedforsequencingdideoxyroibonuccleotidetripphosphate)
C. whenaddNTPisincorporatedintoagrowingDNAstranditpreventsfurther
elongationoftheDNAstrand
1. thereisno3OHonwhichtoaddthenextnucleotide
2. strandlengthcanbeSETonwherethestoopoccurs
D. sequencihatparontngusuallyinvolves4polymerizationmixtures
1. eachpaticularmixturehaslabeledprimersthatallowDNAvisualization
2. eachmixturehasaDNApolymeraseandmultiplecopiesofsingle
strandedtemplateDNA
3. eatwasrunixturethachmixturealsohasall4normaldNTPs
4. themixturesdifferintheddNTP[onewillbeddATP,andddGTP,andone
ddTTP]
5. attheend,eachmixturewillhavenewsynthesizedDNAstrandsineach
mixturebutineachmixtureyouknowtheddNTPisatthe3endofthe
strand
E. thelabeledmixturesarethenthenrunonagelandseparatedbysize
F. thegelisreadfromshortesttolongestfragments
1. aletterisassignedbasedontheddNTPthatwasusedforthemixturethat
wasrunonthatparticulargellane
XIX. RFLPs
A. restrictionfragmentlengthpolymorphisms
B. DNAiscutbyrestrictionenzymesandrunonagelproducesdistinguishableDNA
bands
C. sequencingdifferencesbetweenDNAsamplesresultsindifferentbands
D. soifwehavetheconnectsrestrictionenzymestheRFLPcanbeusedasaDNA
fingerprintsforaperson
E. DNAsequencingisthemostreliablemeansofidentificationandasitbecomes
cheaperitisreplacingsomeusesofRFLP
F. RFLPwillalwaysbefasterandcheaperthansequencingandisstillheavilyused
XX. Applicationsofgeneticengineering
A. drugmanufacture
1. manygneproductshavemedicaluses
a) ahormone,protein,orenzymecanbemadeinbulk
b) humaninsulingenehasbeenmadeinEcoliforover2decades
c) humangrowthhormone
2. medicalmodels
a) transgenicmiceformodelingdiseases
3. genetherapy
a) virusescandelivergenesorDNAintothecellsofhumans
4. improvedfoodsources
a) ricehasbeenengineeredtomakebetacarotenetohelpvitaminA
deficiency[goldenrice]
b) insectresistantplantscotton,corn,etctoexpressabacterialgene
thatcodesforatoxinthatkillsinsects
c) potentialmisusesarealwaysaconcern
d) guidelinesandregulationshavebeenputinplacetohopefully
preventthingssuchasasupervirus,superbacteria,orsuper
weedthatmightbecomeamedicalorecologicaldisaster
XXI. Singlenucleotidepolymorphisms[SNPs]orsnips
A. snipsareDNAsequencevariationsthathappenwhenasinglenucleotideinthe
genomesequenceisaltered
B. Example:AAGGCTAAcouldbechangedtoATGGCTAA
C. foravariationtobeconsideredaSnip,ithastooccurinatleast1%ofthe
population
D. snipsmakeupabout90%ofallhumangeneticvariation,andoccurevery100
300basesalongthe3billionbasehumangenome
E. twoofevery3snipsinvolvereplacementofcytokineswiththymine
F. SNPscanoccurinbothcodingandnoncodingregions
G. donotcausediseasebuttheycanhelpdeterminethelikelihoodthatsomeonewill
developaparticulardisease
H. oneofthegenesassociatedwithAlzheimersiscalledapolipoproteineorApoE
1. thisgenecontains2SNPsthatresultin3possiblealleles
a) E2
b) E3
c) E4
2. eachallelediffersbyoneDNAbaseandtheproteinproducteachgene
differsbyoneaminoacid
3. researchhasshownthatanindividualwhoinheritsatleast1E4allelehas
agreaterchanceofgettingalzheimer's.
4. apparently,thechangeofoneaminoacidintheE4proteinaltersits
structureandfunctionenoughtomakediseasedevelopmentmorelikely
5. theE2alleleseemstoindicateanindividualwhoislesslikelytodevelop
alzheimers
6. SNPsarenotabsoluteindicatorsofdiseasedevelopment
7. ifapersoninherits2E4alleleshestillmayneverdevelopalzheimer's
whileanotherwhohas2E2allelesdoes
8. ApoEisjustonegenethathasbeenlinkedtoAlzheimers
9. likeotherdiseasessuchasheartdisease,diabetes,orcancer
Alzheimerscanbecausedbyvariationsinseveralgenesand
environmentalfactors
10. thepolygeneticnatureofthesediseasesiswhatmakesgenetictestingfor
themsohard
XXII. Embryonicstemcells
A. stemcellsthathavenotyetdifferentiatedintospecificcelltypes
B. foundinpreembryostage
C. theyarepluripotent
1. havingthepotentialtodevelopintoanytypeofbodycell
D. harvestedfromablastocyst
1. destroystheblastocyst
2. coaxedintodevelopingintospecifictypesofcells
E. recentresearchhasfoundsomeadultstemcellsarepluripotent
F. buttheembryonicstemcellsarepluripotenttoagreaterdegree
1. itmaymeanthattheycanbeturnedintoagreaternumberofcelltypes
andthusmaybemoreusefulintreatingdiseasethanadultstemcells
XXIII. Spareembryosvsresearchembryos
A. Spareembryo
1. unusedembryosleftoverfromIVF
2. about2000exist[frozeninnitrogen]
B. researchembryo
1. createdspecificallyforresearchwiththeforeknowledgethattheywillbe
destroyed
XXIV. Whynotconductallresearchonspareembryosratherthancreatenewembryosforthe
purposeofdestroyingthem?
A. thereisagreaterincidenceofgeneticabnormalitywithspareembryos
B. theadvantageoftakingthecellsfromanembryoclonedfromapatientisthatnow
thestemcellswillbegeneticallyidenticaltothepatientandlesslikelytobe
rejected
XXV. IsthereadifferencebetweenusingspareembryosandusingresearchembryosinESC
research?
A. Bydiscardinghundredsofembryostogetonestemcellline,theresearches
abusehumanlife
1. quotebyRichardDoerflingerwiththeU.S.ConferenceofCatholic
Bishops
B. researchesledbyShinyaYamanakahavedevelopedatechniquefor
reprogrammingskinscellstobehaveasiftheywereembryonicstemcells
1. firstdoneinmicein2006andtheninhumansin2007
2. cellsarecalledinducedpluripotentstemcellsoriPScells
3. wemayeventuallybeabletoderiveallthebenefitsoftherapeuticcloning
withouthavingtodestroyhumanembryos
XXVI. Genetherapy
A. definedasthecorrectionofageneticdeficiencybytheadditionofnewDNAtothe
cell
B. hasbeenrecentlyexpandedtoincludetreatmentsofacquireddiseases
C. somaticgenetherapyistheintroductionofgenesintosomatictissues
1. currentdiseasesthatarebeingresearchedinclinicaltrialsarecystic
fibrosis,musculardystrophy,adenosine,deaminasedeficiency,familial
hypercholesterolemia,cancer,AIDS
2. deliverytechniquesforgettingintothecells
a) exvivo
(1) removecellsfromselecttissuesandexposethemtogene
transfervectorsandthenreturnthecellstothepatient
b) invivo
(1) delivergenetransfervectorstobodybyinjectionor
inhalationandhopefullythevectoristargetedtothecorrect
tissuetodeliverthecorrectgene
c) deliveryvectors
(1) viruses
(a) dependingonthevirusused,itwilleitherintegrate
intothechromosomeorsimplybemaintainedinthe
nucleusoritmayonlyremaintransiently
(2) nonviral
(a) liposomes
(i) small,lipidspheres
(ii) lipoplexes[morecomplexwithionic
complexes
(iii) canputDNAinsidethem
(b) Pros
(i) nonviralthereforethereisnoinfectiousrisk
andnoimmuneresponse
(c) Cons
(i) deliveryisnotasefficient
(3) NakedDNA
(a) surprisinglyjustaseffectaslipoplexes
XXVII. RepetitivesequencesofDNAareusefulasgeneticmarkers
A. therearemanyDNAsequencesofunknownfunctionthatarerepeatedhundreds
orthousandsoftimesinthegenome
B. somearescatteredthroughoutgenome
C. somearerepeatedintandemandmayoccuratonlyafewsites
D. atartandemsite,thenumberofrepeatsvarieswidelyinthepopulation
E. therfore,eachdifferentrepeatnumbercanbetreatedasahighlypolymorphicsite
withmultiplealleles
F. thesesitesarecalledVNTRs[variablenumberoftandemrepeats]
G. alsoknownasSTRs
H. minisatellites
1. havearepeatingunitof100to200
a) arestrictionenzymethatcleavesoneithersideofVNTRregion
generatesfragmentsthatdifferinlength
b) thesefragmentsareallelesaredetectedbySouthernBlotsusing
probesthatbindtotherepeatingunit
2. Microsatellites
a) haverepeatingunitsof2to4
(1) becausetheoveralllengthoftheVNTRregionissmall,
PCRcanbeusedtoamplifytheentireregionbyselecting
primersthatflanktheVNTRregion
(2) theresultingfragmentsareseparatedbyelectrophoresis
anddonotrequireprobesfordetection