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Samenvatting Gentechnologie I deel II, BCBT
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samenvatting van 2de deel van gentechnologie deel II door prof. berckx (voor goedkopere prijs stuur via messenger)
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SAMENVATTINGGENTECHNOLOGIE I:
DEEL II
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Abstract
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Jarne Winderickx
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,INHOUD
1. VECTOREN.................................................................................................3
1.1 INLEIDING..................................................................................................................... 3
1.1.1 Bereiding van het te kloneren DNA.....................................................................3
1.1.2 Invoeren van het DNA fragment in gekozen vector............................................3
1.1.3 Inbrengen in de gastheer (GH)...........................................................................3
1.2 VECTOREN.................................................................................................................... 4
1.2.1 Plasmiden........................................................................................................... 4
1.2.1.1 Het plasmide ColE1 als modelsysteem voor de latere ontwikkeling van
kloneringsvectoren................................................................................................................. 4
1.2.1.2 Plasmiden als kloneringsvectoren............................................................................... 5
1.2.1.3 Voorbeelden............................................................................................................... 5
1.2.1.4 Plasmide transformatie in bacteriën........................................................................... 6
Chemische behandeling..................................................................................................... 6
Electroporatie..................................................................................................................... 6
1.2.2 Enkelstrengige DNA bacteriofagen.....................................................................7
1.2.3 Faagmiden.......................................................................................................... 7
1.2.4 Faagvectoren afgeleid van bacteriofaag.............................................................7
1.2.5 Cosmiden............................................................................................................ 9
1.2.8 Intermezzo: verkorten van gDNA......................................................................10
1.2.9 Supervectoren: YACs en BACs...........................................................................10
1.2.10 Keuze van de vector.......................................................................................11
1.2.10.1 Expressievectoren.................................................................................................. 11
1.2.10.2 Constructie van een genomische bank...................................................................11
1.3 NIEUWE GENERATIE KLONERINGSMETHODEN.......................................................................11
1.3.1 Klassieke klonering........................................................................................... 11
1.3.2 TOPO klonering: moderne kloneringen.............................................................12
1.3.2.1 TOPO TA klonering.................................................................................................... 12
1.3.2.2 Zero blund TOPO klonering....................................................................................... 12
1.3.3 Gateway klonering............................................................................................12
1.3.4 Creator klonering.............................................................................................. 14
1.4 OEFENING: NAADLOOS KLONEREN EN GENFUSIE..................................................................14
2. DNA LABELEN........................................................................................... 15
2.1 RADIOACTIEVE MERKERS................................................................................................ 15
2.1.1 Detectie............................................................................................................ 16
2.2 NIET-RADIOACTIEVE MERKERS......................................................................................... 17
2.3 ENZYMATISCHE METHODES VOOR NUCLEÏNEZUUR LABELEN....................................................18
2.3.1 5’-eindstandige merking...................................................................................18
2.3.2 3’-eindstandige merking...................................................................................18
2.3.3 Inwendige merking...........................................................................................18
2.3.4 Merking RNA moleculen....................................................................................19
2.4 CHEMISCHE SYNTHESE VAN NUCLEÏNEZUREN / PRIMERS / OLIGONUCLEOTIDEN...........................19
2.5 CHEMISCHE OF FOTOMERKING VAN NUCLEÏNEZUREN............................................................20
3. TRANSFORMATIE VAN BACTERIËN EN SELECTIE VAN TRANSFORMANTEN......20
3.1 TRANSFORMATIE........................................................................................................... 21
3.2 SELECTIE MET BEHULP VAN SELECTIE-MERKERS...................................................................21
3.3 TRANSFORMATIE-EFFICIËNTIE.......................................................................................... 21
3.4 KOLONIEHYBRIDISATIE................................................................................................... 21
4. DNA SEQUENERINGS METHODES................................................................22
4.1 SANGER METHODE / DIDEOXY SEQUENERING......................................................................22
4.2 SEMI-AUTOMATED SEQUENERING.....................................................................................22
4.3 SEQUENEREN VAN COMPLETE GENOMEN: SHOTGUN SEQUENERING.........................................23
1
, 4.4 CAPILLAIRE ELEKTROFORESE........................................................................................... 24
4.5 BLAST / BASIC LOCAL ALIGNMENT SEARCH TOOL: OVERSLAAN..............................................25
4.6 NEXT GENERATION SEQUENCING / NTG SEQUENCING..........................................................25
4.6.1 Pyrosequencing................................................................................................26
4.6.2 Illumina sequencing..........................................................................................27
4.6.3 SOLID sequencing / sequencing by oligoligation and detection........................28
4.6.4 Enkelvoudige DNA molc sequenering / Helicos sequenering.............................29
4.6.5 SMRT sequenering............................................................................................30
4.6.6 Isolatie DNA van interesse................................................................................30
4.6.7 ChIP sequencing...............................................................................................31
4.6.8 Ion Torrent sequencing.....................................................................................31
5. GENEXPRESSIE.........................................................................................32
5.1 HETEROLOGE / RECOMBINANTE GENEXPRESSIE IN PROKARYOTEN............................................32
5.1.1 Induceerbare promotor voor genexpressie.......................................................32
5.1.1.1 Trp operon................................................................................................................ 33
5.1.1.2 Lac-operon............................................................................................................... 33
5.1.1.3 Tac-promotor: hybride Trp en lac promotor...............................................................34
5.1.1.4 pL promotor.............................................................................................................. 34
5.1.1.5 T7 gen 10 promotor.................................................................................................. 34
5.1.1.6 Regeling ProU operon / osmoregulatie......................................................................35
5.1.1.7 Laboschaal vs industrieel......................................................................................... 35
5.1.2 Fusie-eiwitten................................................................................................... 36
5.1.3 Faag-display..................................................................................................... 36
5.1.3.1 faag-display van AL-fragmenten............................................................................... 37
5.1.3.2 VHH AL / nanobodies / Heavy-chain only AL.............................................................40
5.1.4 Controle eiwitsynthese.....................................................................................40
5.1.4.1 Codon gebruik: aanpassen aan organisme...............................................................40
5.1.4.2 N-terminale AZ bepalen eiwitstabiliteit mee.............................................................40
5.1.4.3 Eiwitopvouwing bevorderen: S-S brug vorming.........................................................41
5.1.5 Verwijderen van ABR-genen..............................................................................41
5.2 VOORBEELDEN VAN RECOMBINANTE THERAPEUTISCHE EIWITTEN.............................................42
5.2.1 mens IFN-beta klonering en expressie..............................................................42
5.2.2 menselijk groeihormoon / MGH klonering.........................................................43
5.2.3 Biologische inperking van genetisch veranderde L. lactis.................................44
5.2.4 Recombinant influenzavaccin aangemaakt in E.coli.........................................45
2
, 1. VECTOREN
1.1 INLEIDING
Kloneren = invoeren DNA segment (uit bacterie, plant, dier,…) in geschikte
dragermolecule / vector om daarna stabiel te doen vermenigvuldigen in
gekozen GH
o Vector bepaald door GH + moet kunnen worden doorgegeven aan
volgende generaties
Stappen kloneringen: bereiden DNA-fragment invoeren in vector
inbrengen in GH zoeken naar GH die vector hebben opgenomen (/
positieve klonen) amplificeren
o Hoe amplificeren? klonaal uitgroeien (= alle bacteriën op
agarplaat doen groeien verdunnen isolatie 1 kolonie
monocultuur
1.1.1 BEREIDING VAN HET TE KLONEREN DNA
= nieuw DNA (PCR-product, DNA-fragmenten na restrictiedigest,…) als in
vivo geïsoleerd DNA (gDNA / genomisch DNA)
Ook onderscheid: klonering 1 enkel fragment of gehele mengsels
fragmenten
o Gehele mengsels: om cDNA en gDNA banken te maken
Hoe? cDNA’s van alle voorkomende mRNA in cel kloneren
1.1.2 INVOEREN VAN HET DNA FRAGMENT IN GEKOZEN VECTOR
Oorspronkelijk: ligatiereactie tussen insert + vector (= klassieke
klonering)
1.1.3 INBRENGEN IN DE GASTHEER (GH)
Transformatie vs transfectie: vreemd DNA binnenbrengen in virussen,
bacteriën,.. vs vreemd DNA binnenbrengen in eukaryote cellen
3
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