Summary
Samenvatting Moleculaire biologie en DNA technologie - MLT2 - Hogent
- Course
- Institution
Samenvatting van de slides en syllabus waar een syllabus beschikbaar was.
[Show more]
1 review
By: lenalauwrensens • 1 year ago
Seller
Follow
mltmdk
Reviews received
Content preview
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:
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller mltmdk. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $9.16. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
73918 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now