Samenvatting Biotechnologie & immunologie. In de samenvatting worden de volgende aspecten behandeld: restrictie enzymen, sticky ends, Modification enzymes, DNA replicatie in vitro (PCR), Molecular cloning, plasmides, reporter genes, gene fusions, Operon fusions, Protein fusions, plasmides als cloni...
Biotechnologie & Gezondheid
11.1
Genetc enginee ing: efe s to the sse of in iit o techniqses to alte genes in the la..
DNA .e isolated in specifc f aggents ps ifed fo fs the ganipslaton.
Rest icton enzyges: can chegically godify DNA.
Recognise specifc .ase seqsences within DNA and cst the phosphodieste .acb.one.
Ressltng in dos.lesst anded . eabs.
o P otect p oba yotes f og hostne fo eign DNA (ii ss)
o In iit o ganipslaton
o Genetc enginee ing
Mechanism of restricton enzymes.
Rest icton endonscleases a e diiided into th ee gajo classes.
o Type 1 en 3 est icton enzyges .ind tot he DNA at thei ecogniton seqsences .st
cst the DNA at soge distance away.
o Type 2 est icton enzyges cleaie the DNA within thei ecogniton seqsences,
gabing this class of enzyges gsch go e ssefsl fo t he specifc ganipslaton of DNA.
Most of the DNA seqsences ecognized .y type 2 est icton enzyges a e sho t inie ted
epeats of 4 to 8 .ase pai s.
EcoRI bnipt in de seqsence en e .lijien stcby ends oie
EcoRV bnipt in de seqsence en e .lijien .lsnt ends oie
F aggents with stcby ends a e .enefcial fo golecsla cloning of DNA.
Methyl g osps a e added .y these enzyges to p otect the est icton site f og cstng .y
EcoRI/RV.
Modifcaton: Protecton from restricton.
Modifcaton enzyge: enzyge that chegically alte s .ases within a est icton enzyge
ecogniton site and thss p eients the site f og .eing cst.
(a cell gsst p otect its own DNA f og inadie tent dest scton .y its own est icton
enzyges)
Gel electrophoresis: Separaton of DNA molecules
Gel elect opho esis: techniqse fo sepa aton of nscleic acid golecsles .y passing an elect ic
cs ent th osgh a gel gade of aga ose o polyac ylagide
o Van negatef naa positef. (fosfaatg oepen zijn negatef geladen)
o Sgall o cogpact golecsles gig ate go e apidly than la ge golecsles.
o The highe the concent aton of aga ose in the gel, the g eate is the esistance to
goiegent fo la ge golecsles.
1
,11.2
Denats ed DNA: the 2 st ands a e sepa ated
The single st and scan fo g hy. id dos.lesst anded golecsles with othe singles
st anded DNA golecsles .y cogplegenta y .ase pai ing.
= hy. idizaton
Used in detectng, cha acte izing, and identfying seggents of DNA en RNA.
Seggents of singlesst anded nscleic acids whose identty is al eady bnown and that a e ssed
in hy. idizaton a e called: nscleic acid p o.es (p o.es)
Can .e gade adioactie o la.eled with chegicals that a e colo ed o yield
fso escent p odscts.
Southern and northern blots
o Sosthe n .lotng: p o.es of bnown seqsence a e hy. idized to ta get DNA
f aggents that haie .een sepa ated .y gel elect opho esis.
Sosthe n .lot: The hy. idizaton p oceds e in which DNA is the ta get seqsence in
the gel, and RNA o DNA is the p o.e.
No the n .lot: sses RNA as the ta get seqsence and DNA o RNA as the p o.e to
detect gene exp ession. (gRNA)
Hy. idizaton can .e detected .y gonito ing the la.eled p o.e that has .osnd tot he
geg. ane.
11.3
PCR (polyge ase chain eacton): is essentally DNA eplicaton in iit o.
Can copy seggents of DNA .y sp to a .illionfold in the test ts.e = agplifcaton
Uses the enzyge DNA polyge ase copies DNA golecsles
Stappen:
1. Tegplate DNA is denats ed .y heatng.
2. Two a tfcial DNA oligonscleotde p ige s fanbing the ta get DNA on each st and a e
added in excess. This enss es that gost tegplate st ands anneal to a p ige , and not
to each othe , as the gixts e cools.
3. DNA polyge ase then extends the p ige s ssing the o iginal DNA as the tegplate.
4. Afte an app op iate incs.aton pe iod, the gixts e is heated again to sepa ate the
st ands, .st now the ta get gene is p esent in twice the o iginal agosnt. The gixts e
is then cooled to allow the p ige s to hy. idize with cogplegenta y egions of newly
synthesized DNA, and the whole p ocess is epeated.
PCR & Polymerases
A the gosta.le DNA polyge ase isolated f og the The gss aqsatcss (.acte ie) is ssed
.ecasse high tegpe ats es a e ssed t odenats e the dos.lesst anded copies of DNA in iit o.
DNA polyge ase f og T. aqsatcss is called Taq polyge ase (95 g aden)
DNA polyge ase f og Py ococcss fs iosss is called Pfs polyge ase (100 g aden)
2
,PCR applicatons
Reverse transcripton (RTsPCR) can .e ssed to gabe DNA f og an gRNA tegplate.
Used to detect if a gene is exp essed o to p odsce an int onsf ee esba yotc gene fo
exp ession in .acte ia as desc i.ed fo the ho gones insslin and sogatot opin.
Reverse transcriptase: conie t RNA into cogplegenta y DNA (cDNA)
To qsantfy the agosnt of inital ta get DNA o RNA in a sagple, a p oceds e called
qsanttatie PCR (qPCR) can also .e ssed. (fso escent)
11.4
Molecular cloning: a f aggent of DNA is isolated and eplicated.
Isolate the desi ed gene f og its o iginal locaton and goie it to a sgall, sigple, and
ganipsla.le genetc elegent, ssch as a plasgid o ii ss, which is called a vector.
Resslts in recombinant DNA = a DNA golecsle that contains DNA f og two o go e
sos ces.
Keys:
o Rest icton enzyge
o DNA ligase
o PCR
o Synthetc DNA
Steps in Gene cloning
1. Isolaton and f aggentaton of the sos ce DNA.
2. Inse tng the DNA f aggent into a cloning iecto .
(gost iecto s (plasgids o ii ssses) a e typically designed to allow inse ton of
fo eign DNA at a est icton site that csts the iecto withost afectng its eplicaton)
If the sos ce DNA is PCRsgene ated, DNA ligase is ssed to joint he agplifed DNA to
specialized iecto s.
3. Int odscton of the cloned DNA into a host o ganisg.
Genogic li. a y: gany dife ent clones can .e ps ifed f og the gixts e, each
containing dife ent cloned DNA seggents f og the sos ce o ganisg.
Shotgsn cloning: gabing a genogic li. a y .y cloning andog f aggents of a
genoge.
Detectng proteins expressed in the cloning host
Ant.odies can .e ssed to detect a p otein of inte est. Ant.odies a e p oteins of the
iggsne systeg that .ind in a highly specifc way to a ta get golecsle, the antgen. In this
case the p otein encoded .y the cloned gene is the antgen. Becasse ie y litle of the antgen
is p esent in each colony, only a sgall agosnt of ant.ody is .osnd, and so a highly sensitie
p oceds e fo detectng .osnd ant.ody gsst .e ssed. In p actse, adioisotopes,
fso escent chegicals, o enzyges a e tssed.
3
, 11.5
Sitesdi ected gstagenesis: sses synthetc DNA plss DNA cloning techniqses to int odsce
gstatons into genes at p ecisely dete gined sites.
Synthesizing DNA
Once the oligonscleotde is the desi ed length, it is cleaied f og the solid phase ssppo t .y a
specifc eagent and ps ifed to eliginate .yp odscts and contaginants.
Site-directed mutagenesis
By alte ing gene seqsences to p odsce agino acid seqsence changes, sitesdi ected
gstagenesis is ssed to ganipslate p otein cha acte istcs ssch as enzyge actiity o p oteins
.inding afnity.
1. Clone into plasgid and denats e
2. Add synthetc oligonycleotde with one .ase gisgatch
3. Extend single st and with DNA polyge ase
4. T ansfo gaton and selecton
Sogatot opin=g oei ho goon
Casete mutagenesis and gene disrupton
DNA casetes (synthetc f aggents): can .e ssed to gstate DNA in a p ocess bnown as
cassete gstagenesis.
Anothe type of casete gstagenesis is gene dis spton.
Casetes a e inse ted into the giddle of a gene, thss dis sptng the coding seqsence.
11.6
Reporter Genes
Repo te gene: it encodes a p otein that is easy to detect and assay.
The f st gene to. e ssed widely as a epo te was the Esche ichia coli gene lac,, which
encodes the enzyge Bsgalactosidase, eqsi ed fo lactose cata.olisg.
The g een fso escent p otein (GFP) is widely ssed as a epo te .
May .e exp essed in gost cells as it is sta.le and casses litle o no dis spton of host
cell geta.olisg.
Gene fusions
Gene fssions: consist of seggents f og two dife ent genes.
2 types
o Operon fusions: a coding seqsence that etains its own t anslatonal sta t site and
signals is fssed to the t ansc iptonal signals of anothe gene.
o Protein fusions: genes that encode two dife ent p oteins a e fssed togethe so that
they sha e the sage t ansc iptonal and t anslatonal sta t and stop signals.
4
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