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Samenvatting Moleculaire biologie en DNA technologie - MLT2 - Hogent
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InhoudH1. Isolatie van nucleïnezuren .......................................................................................................................................... 5
1.1. Leerdoelen ........................................................................................................................................................ 5
1.2. Isolatie van nucleïnezuren ................................................................................................................................ 5
1. Hoe kunnen we cellen openbreken om er DNA uit te isoleren? ...................................................................... 5
2. Hoe zuiveren we het DNA? Hoe verwijderen we de overige componenten? ...................................................... 6
3. Hoe bewaren we het geïsoleerd DNA? ................................................................................................................. 7
4. Hoeveel DNA we kunnen we isoleren uit een bepaald monster/staal? Hoe bew aren we startmateriaal? ..... 7
1.3. Kwaliteitscontrole van geïsoleerd DNA en/of RNA ........................................................................................... 7
1.4. Extractierobots .................................................................................................................................................. 7
1.5. Remmende factoren van de PCR ...................................................................................................................... 7
H2. PCR-analyse ................................................................................................................................................................ 8
2.1. Leerdoelen ............................................................................................................................................................. 8
2.2. Basisprincipes PCR ................................................................................................................................................. 8
2.2.1. Begrippen ........................................................................................................................................................ 8
2.2.2. Doel van PCR ................................................................................................................................................... 9
2.2.3. Principe van PCR ............................................................................................................................................. 9
2.3. Fasen PCR-reactie .................................................................................................................................................. 9
1. Denaturatie fase (95°C) ......................................................................................................................................... 9
2. Annealing fase (45°C – 60°C) ................................................................................................................................. 9
3. Extensiefase (72°C)................................................................................................................................................ 9
2.4. Berekeningen PCR-mix ......................................................................................................................................... 10
2.4.1. Verdunnen template DNA............................................................................................................................. 10
2.4.2. Primeroplossingen ........................................................................................................................................ 10
2.4.3. Mastermix (MM) ........................................................................................................................................... 10
2.5. DNA-contaminatie/PCR-contaminatie ................................................................................................................. 10
Bron van contaminatie ............................................................................................................................................ 10
alle nucleïnezuur bevattende materialen in labo ........................................................................................... 10
Hoe verspreid? ........................................................................................................................................................ 10
Hoe veroorzaakt? .................................................................................................................................................... 10
Hoe voorkomen? ..................................................................................................................................................... 11
Infrastructuur pré- en post-PCR labs ...................................................................................................................... 11
2.6. PCR-controles ....................................................................................................................................................... 12
2.6.1. Negatieve controles ...................................................................................................................................... 12
2.6.2. Positieve controles ........................................................................................................................................ 12
2.6.3. Interne controles........................................................................................................................................... 12
2.6.4. Analyse PCR-fragmenten .............................................................................................................................. 12
2.7. PCR-componenten ............................................................................................................................................... 13
2.7.1. dNTP’s ........................................................................................................................................................... 13
, 2.7.2. primers .......................................................................................................................................................... 13
2.7.3. Polymerase ................................................................................................................................................... 14
2.7.4. MgCl2 buffer ................................................................................................................................................. 14
2.7.5. hulpstoffen .................................................................................................................................................... 14
2.8. PCR-toepassingen ................................................................................................................................................ 14
H3. Scheiding en detectie van DNA ................................................................................................................................ 15
3.1. Leerdoelen ........................................................................................................................................................... 15
3.2. Begrippen ............................................................................................................................................................. 15
3.3. Principe elektroforese .......................................................................................................................................... 15
3.4. Agarosegelelektroforese (AEG) ............................................................................................................................ 15
Uitvoering................................................................................................................................................................ 15
Scheidend vermogen en resolutie .......................................................................................................................... 16
Voorbereiding monsters ......................................................................................................................................... 16
Moleculaire marker als referentiepunt................................................................................................................... 16
Migratie en detectie van nucleïnezuren ................................................................................................................. 16
Samenvatting agarosegelelektroforese (AEG) ........................................................................................................ 17
3.5. Polyacrylamide gelelektroforese (PAGE) ............................................................................................................. 18
Toepassing .............................................................................................................................................................. 18
Resolutie en scheidend vermogen .......................................................................................................................... 18
3.6. Capillaire gelelektroforese (CE) ............................................................................................................................ 18
Principe ................................................................................................................................................................... 18
SAMENVATTING .......................................................................................................................................................... 18
H4. De stroom van genetische informatie ...................................................................................................................... 19
4.1. Moleculaire bouwstenen van leven ..................................................................................................................... 19
DNA (desoxyribonucleïnezuur) ............................................................................................................................... 19
RNA (Ribonucleïnezuur) .......................................................................................................................................... 20
4.2. Het genoom – blauwdruk voor leven.................................................................................................................. 20
4.2.1. Behoud en doorgeven van informatie .......................................................................................................... 20
4.2.2. Het genoom .................................................................................................................................................. 20
4.3. Genexpressie – omzetting genetische informatie tot lichaamseigenschappen .................................................. 22
HET CENTRALE DOGMA .......................................................................................................................................... 22
GEMODIFCIEERDE VERSIE DOGMA ......................................................................................................................... 23
4.4. Regulatie van genexpressie – differentiatie en modelorganismen ..................................................................... 25
4.4.1. Genexpressie op verschillende niveaus ........................................................................................................ 25
................................................................................................................................................................................. 25
4.4.2. Verband differentiatie en genexpressie........................................................................................................ 25
4.4.3. Verband fenotypische plasticiteit en genexpressie ...................................................................................... 26
4.4.4. Prokaryoten vs. Eukaryoten .......................................................................................................................... 26
4.5. Genexpressie en de architectuur van de cel ........................................................................................................ 26
, 4.5.1. Compartimentalisatie ................................................................................................................................... 26
4.5.2. Prokaryoten vs. eukaryoten .......................................................................................................................... 26
4.6. Evolutie van het genoom ..................................................................................................................................... 27
4.6.1. Diversiteit van genomen ............................................................................................................................... 27
4.6.2. Variaties en mutaties .................................................................................................................................... 27
4.6.3. Genduplicaties en genfamilies ...................................................................................................................... 28
4.7. Onderzoeksgebieden ........................................................................................................................................... 28
H5. Genexpressie ............................................................................................................................................................ 29
5.1. Dogma moleculaire biologie ................................................................................................................................ 29
5.2. van DNA tot RNA: transcriptie ............................................................................................................................. 29
5.2.1. verschillen tussen DNA en RNA..................................................................................................................... 29
5.2.2. Efficiëntie van eiwitsynthese ........................................................................................................................ 29
5.2.3. RNA-polymerase ........................................................................................................................................... 30
5.2.4. Verschillende soorten RNA ........................................................................................................................... 30
5.2.5. Binding – initiatie – elongatie – terminatie transcriptie ............................................................................... 31
5.2.6. RNA-processing van eukaryotische mRNAs in de nucleus ............................................................................ 33
5.2.7. Transcriptie unit ............................................................................................................................................ 35
5.3. van RNA tot eiwit: translatie ................................................................................................................................ 36
Basis transport van mRNA naar cytosol .................................................................................................................. 36
tRNA met anticodon................................................................................................................................................ 36
Translatie met mRNA-molecule ............................................................................................................................. 36
Polyribosoom of polysomen ................................................................................................................................... 39
Post-translationele modificaties ............................................................................................................................. 39
Gecontroleerde afbraak van eiwitten ..................................................................................................................... 39
5.4. van DNA naar eiwit .............................................................................................................................................. 40
H6. Regulatie genexpressie ............................................................................................................................................. 41
6.1. Situering en inleiding ........................................................................................................................................... 41
Structuur mRNA prokaryoot vs. eukaryoot ............................................................................................................ 41
6.2. Regulatie van genexpressie bij prokaryoten ........................................................................................................ 42
6.3. Regulatie van de genexpressie bij eukaryoten .................................................................................................... 44
6.3.1. Chromatine ................................................................................................................................................... 45
6.3.2. Transcriptiefactoren...................................................................................................................................... 46
6.3.3. RNA-processing ............................................................................................................................................. 46
6.3.4. Regulatorische niet-coderende RNA’s .......................................................................................................... 46
H7. Epigenetica ............................................................................................................................................................... 48
7.1. Genexpressie en chromatine (+H6) ..................................................................................................................... 48
7.2. Gen dosage........................................................................................................................................................... 49
7.3. Genomic imprinting ............................................................................................................................................. 49
7.3.1. Genetische ziekten t.g.v. genomic imprinting .............................................................................................. 50
, 7.4. RNA-gebaseerde mechanismen voor ‘gene silencing’ ......................................................................................... 50
7.5. Regenereren en herprogrammeren ..................................................................................................................... 50
7.6. Epigenetica en kanker .......................................................................................................................................... 50
7.6. Epigenetica en milieu ........................................................................................................................................... 51
H8. Erfelijkheid en kanker ............................................................................................................................................... 52
8.1. Ontstaan van kanker ............................................................................................................................................ 52
8.1.1. Kanker: een ziekte van de cel........................................................................................................................ 52
8.2. Kanker als meerstappenproces ............................................................................................................................ 53
8.3. Verstoring van celdelingsproces – verworven eigenschappen van kankercellen................................................ 54
8.4. Proto-oncogenen en tumorsuppressorgenen ..................................................................................................... 55
8.4.1. Proto-oncogenen tot oncogenen .................................................................................................................. 55
8.5. DNA-herstelgenen/8.6. Genetische instabiliteit/8.7. Virussen /8.8. Epigenetica en kanker (H7.5) .................... 56
8.9. Kanker en erfelijkheid .......................................................................................................................................... 56
8.10. Kankertherapie + oefening dia 62 ............................................................................................................. 57
H9. Oefeningen practicum .............................................................................................................................................. 57
H10. Technologie ............................................................................................................................................................ 58
10.1 Modelorganismen ............................................................................................................................................... 58
10.2. Kweken van eukaryote cellen ............................................................................................................................ 58
10.3. Amplificatie van DNA en RNA ............................................................................................................................ 58
10.3.1. PCR .............................................................................................................................................................. 58
10.3.2. reverse transcriptie ..................................................................................................................................... 58
10.3.3. kloneren ...................................................................................................................................................... 58
10.3.4. qPCR ............................................................................................................................................................ 60
10.4. Sequentiebepaling ............................................................................................................................................. 62
10.5. Blotting ............................................................................................................................................................... 62
10.6. Hybridisatie en probes ....................................................................................................................................... 64
10.6.1. Denaturatie, renaturatie en hybridisatie .................................................................................................... 64
10.6.2. Probes ......................................................................................................................................................... 65
10.6.3. Hybridisatie ................................................................................................................................................. 66
, Moleculaire biologie en DNA-technologie
H1. Isolatie van nucleïnezuren Tip: test jezelfs en leerdoel dia’s op ppt
1.1. Leerdoelen
1.2. Isolatie van nucleïnezuren
• Doel → Isoleren (= opzuiveren) van DNA uit cellen/weefsels
• Belang → Enzymatische ‘down stream’; onzuivere DNA stoort enzymwerking
• Principe
o Lyse
o Verwijderen ongewenste componenten
o Bewaren van geïsoleerd DNA
o Kwaliteitscontrole van geïsoleerde DNA (kwalitatief en kwantitatief)
1. Hoe kunnen we cellen openbreken om er DNA uit te isoleren?
Lysemethode → afhankelijk van staaltype (grote diversiteit in cel architectuur)
→ ≠ lysemethoden met ≠ voorbehandelingen
• Chemisch
o Detergens: maakt celmembraan kapot (SDS)
o Alkalische buffer (NaOH)
• Biologisch (enzymen)
o Proteïnase K: breekt EW af uit celmembraan en op de NZ → nucleasen onschadelijk maken
▪ Eigenschappen van proteïnase K: Breed-spectrum proteinase
Knipt na hydrofobe AZ
Temperatuurprofiel
Stabiel over breed pH-bereik
Werking verhoogd door detergenten en chaotropen
→ (denatureren substraat: enzym meer toegang)
▪ Activiteit proteïnase K verhogen Lysozyme: breekt celwand van grampositieven open
Detergenten en chaotrope stoffen
→ (denatureren substraat: enzym meer toegang)
• Fysisch: warmte, osmose
• Mechanisch: vortex, soniceren
• Combinatie
o Humane cellen proteïnase K, detergent, warmte, chaotrope stof/zout (in lysebuffer)
o Bacteriën NaOH en warmte
, 2. Hoe zuiveren we het DNA? Hoe verwijderen we de overige componenten?
Vroeger: Fenol-chloroform extractie
• Fasen: 1. Waterige fase → DNA
2. Interfase → celdebris
3. Organische fase → proteïnen, lipiden
• Extractie: isopropanol precipitatie om DNA te controleren
Voordelen Nadelen
- Zuiver DNA - Tijdrovend
- Zeer goede opbrengst - Isopropanol precipitatie
- Schadelijke organische solventen
- Residuele organische solventen interfereren met
DNA-concentratiebepaling en enzymatisch DNA-
manipulatie
Sinds 1990: Boom-‘extractie’ = kolomchromatografie
1. Adsorptie van DNA op silicagel
• Met behulp van bindingbuffer met een hoge zoutconcentratie en chaotrop agens
• Functie van buffer:
o Verwijderen watermantel van DNA
o Verwijderen watermantel van kolom
o Vormen kationbrug tussen silicakolom en negatief geladen DNA
2. Verwijderen van contaminanten (wassen, flow through)
• Wassen met ethanol (chaotrop) en hoge zoutconcentratie
• DNA blijft gebonden aan kolom
• Contaminanten worden weggewassen
• Verwijderen van enzyminhibatoren
3. Elutie van DNA → door lage zoutconcentratie
Voordelen
- Snel
- Minder schadelijke organische solventen
- Zuiver en geconcentreerd DNA
- Makkelijk te automatiseren
Samenvatting DNA-isolatie technieken:
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