In deze cursus komt de recombinant DNA-technologie via de methode van klassieke klonering aan bod. Het belang en gebruik van type I, II, III en speciale restrictie-enzymen in E. coli wordt kort besproken. Het praktisch gebruik van type II restrictie-enzymen wordt uitvoerig bekeken.
Vervolgens wo...
Mutaties en het ontstaan van kanker ................................................................................................................ 6
Kenmerken tumorcellen ................................................................................................................................ 6
Switch van goedaardige tumor naar kwaadaardige tumor.......................................................................... 7
Verwijderen tumoren is niet mogelijk via chirurgie .................................................................................... 7
Proto-oncogenen, oncogenen en tumorsuppressorgenen.............................................................................. 7
Tumormerkers .......................................................................................................................................... 8
Delingsactiviteit wordt beïnvloed door ...................................................................................................... 8
Wordt kanker overgeërfd? ............................................................................................................................ 9
Uitschakelen van tumorsuppressorgenen ...................................................................................................... 9
Onderscheid sporadisch of niet – overgeërfde vorm en familiaal of overgeërfde vorm ............................... 9
Meerstappentheorie (Multi hit theory)........................................................................................................ 10
Aantal tumorsuppressorgenen ................................................................................................................ 11
Aantal oncogenen ................................................................................................................................... 11
Groeiend belang van tumormerkers ............................................................................................................ 12
FOBT (Fecal Occult Blood test) ................................................................................................................. 13
PAP-test (Papanicolaou test) ................................................................................................................... 13
Technieken voor diagnose van moleculaire merkers ................................................................................ 14
Liquid biopsie.............................................................................................................................................. 14
NIPT test ................................................................................................................................................. 15
Doelgerichte therapieën, nieuwe wapens in de strijd .................................................................................. 15
Monoklonale antilichamen ...................................................................................................................... 16
Tyrosine Kinase Inhibitoren (TKI) ............................................................................................................. 17
Voorbeelden van carcinoma’ s .................................................................................................................... 18
Colorectale tumoren (uitleggen + vragen beantwoorden) ........................................................................ 18
Borsttumoren.......................................................................................................................................... 19
Voorbeeldvragen Kanker................................................................................................................................. 19
Is kanker overerfbaar? Motiveer ................................................................................................................. 19
3 meest voorkomende kankers bij mannen en vrouwen .............................................................................. 19
Hoe diagnose voor tumor/kanker stellen? Manieren om tumoren op te sporen + geef alle mogelijke
tumormerkes .............................................................................................................................................. 20
Therapie voor het bestrijden van kanker ..................................................................................................... 20
Op welk vlak werken de geneesmiddelen .................................................................................................... 21
Welke zijn de jongste generatie veelbelovende geneesmiddelen ................................................................. 21
1
, Hoe werken mAB tegen kankers (4 manieren) ............................................................................................. 21
Wat weet je over een oncogen? Geef voorbeelden ..................................................................................... 22
Wat weet je over een tumorsuppresorgen? Geef voorbeelden .................................................................... 22
2 hit-theorie van Knudson ........................................................................................................................... 22
het ontstaan van kanker/tumor is een meerstapsproces. Leg uit ................................................................. 23
Gebruik van E-coli stammen en restrictie-enzymen in klassieke kloneringsmethoden ...................................... 24
Restrictie-enzymen ..................................................................................................................................... 24
3 types van restrictie enzymen ................................................................................................................ 24
Speciale restrictie-enzymen in E. coli ....................................................................................................... 25
Type II restrictie-enzymen ........................................................................................................................... 25
Modifying enzymen ............................................................................................................................. 26
Types van herkende sequenties................................................................................................................... 26
Te onderscheiden knipwijzen ...................................................................................................................... 28
Blunt eindjes ........................................................................................................................................... 28
Sticky eindjes .......................................................................................................................................... 28
Frequentie van voorkomen van restrictieplaatsen ....................................................................................... 28
Invloed van de omgevende sequentie op de knip efficiëntie ........................................................................ 28
Manieren van knippen van de herkende sequentie...................................................................................... 29
Begrippen: Prototype – Isoschizomeer – neoschizomeer ............................................................................. 29
Invloed van methylatie op het knippen van type II restrictie – enzymen....................................................... 30
Dam en Dcm methylatie .......................................................................................................................... 30
CpG en CpNpG methylatie in eukaryoten ................................................................................................. 31
Gebruik van restrictie – enzymen .................................................................................................................... 31
Definitie Unit .............................................................................................................................................. 31
Buffersamenstelling .................................................................................................................................... 31
Temperatuur............................................................................................................................................... 31
Staractiviteit ............................................................................................................................................... 31
Stoppen van de reactie ............................................................................................................................... 32
Knippen van DNA met restrictie – enzymen ................................................................................................. 32
Opstellen van gecombineerde digesten ................................................................................................... 33
Betekenis symbolen............................................................................................................................. 34
Oefeningen staractiviteit – bufferconcentratie ........................................................................................ 34
Staractiviteit ........................................................................................................................................ 34
Bufferconcentratie .............................................................................................................................. 35
Plasmiden ....................................................................................................................................................... 35
Definitie van een plasmide .......................................................................................................................... 35
Structuur plasmide...................................................................................................................................... 36
2
, Antibiotica ’s ........................................................................................................................................... 37
Prokaryotische promotors ....................................................................................................................... 37
Incompatibiliteit.......................................................................................................................................... 38
Gastheerbereik ........................................................................................................................................... 39
Kopijnummer .............................................................................................................................................. 39
E. coli strengen in het labo .............................................................................................................................. 39
E. coli .......................................................................................................................................................... 39
Constructie van een recombinant plasmide via klonering met T4 DNA ligase ................................................... 39
Definitie basale kloneringsvector................................................................................................................. 39
Kloneren in praktijk ................................................................................................................................. 40
Oefeningen ......................................................................................................................................... 41
Constructie van een recombinant plasmide via klonering met topoimerase ..................................................... 42
De TOPO TA kloneringstechniek .................................................................................................................. 42
Positieve selectie CCdb – gen................................................................................................................... 42
Gibson Assembly Cloning ................................................................................................................................ 43
Principe van de techniek ............................................................................................................................. 43
Toepassing Gibson Assembly – Nucleotiden niveau ................................................................................. 44
............................................................................................................................................................... 44
Golden Gate Cloning ....................................................................................................................................... 45
Doel van het kloneren via gateway .............................................................................................................. 45
BP reactie ................................................................................................................................................... 46
LR reactie .................................................................................................................................................... 46
Synthese en klonering van cDNA ..................................................................................................................... 47
Doel cDNA banken ...................................................................................................................................... 47
RNA bereiding ............................................................................................................................................. 47
Bereiding totaal RNA (Stappen kennen + uitleggen) ................................................................................. 47
Isolatie van de Poly A+ fractie = mRNA ........................................................................................................ 49
Afzonderen van mRNA ............................................................................................................................ 49
Synthese van cDNA ......................................................................................................................................... 50
Smart Race cDNA amplification ................................................................................................................... 50
Klonering van cDNA ........................................................................................................................................ 51
Homopolymeer tailing................................................................................................................................. 51
Adaptors ..................................................................................................................................................... 53
Transformatie ................................................................................................................................................. 54
Screenen van de bank ..................................................................................................................................... 54
Klonering van RNA’s die geen poly – A staart dragen ....................................................................................... 54
RACE of Rapid Amplification of cDNA ends ...................................................................................................... 54
3
, 5’ RACE ....................................................................................................................................................... 55
3’ RACE ....................................................................................................................................................... 56
Toepassing Race of cDNA ends (Kunnen op het examen) ............................................................................. 57
Maak het 5’ en 3’ RACE product en kloneer het in de vector Maak zelf de 5’ RACE en 3’ Race producten en
ligeer ze in onderstaande vector (typische examenvraag) ........................................................................ 57
In vitro gekoppelde transcriptie-translatie systemen ....................................................................................... 59
Merkingstechnieken voor het aangemaakt eiwit ......................................................................................... 60
Expressie van gekloneerde genen in E. coli ...................................................................................................... 61
Waarom expressie in E. coli ......................................................................................................................... 61
Decodering van de genetische informatie: transcriptie ............................................................................ 62
Decodering van de genetische informatie : translatie............................................................................... 62
Decodering van de genetische informatie : secundaire modificaties ......................................................... 63
Algemene opbouw en gewenste eigenschappen van een expressievector ................................................... 63
Een expressievector voor expressie in E. coli ............................................................................................ 63
Toegankelijkheid van het ATG codon van de expressievector ................................................................... 64
5’ sticky knipper; voorbeeld NcoI ......................................................................................................... 64
3’ sticky knipper; voorbeeld Kpn I ........................................................................................................ 64
Toegankelijkheid van het eerste gewenste codon van het mature eiwit in het cDNA ................................ 65
Quick Change Site Directed Mutagenese – PCR (Oefeningen kunnen maken) ............................................... 65
Voorbeeld ............................................................................................................................................... 65
Oefening ............................................................................................................................................. 66
Overzicht van veel gebruikte promotors en hun regulatie................................................................................ 68
Belang van reguleerbaarheid....................................................................................................................... 68
Lek – expressie ........................................................................................................................................ 69
Factoren die de finale opbrengst van een eiwit bepalen........................................................................... 69
pET expression vectors................................................................................................................................ 69
Expressie van recombinant eiwit in Pichia pastoris .......................................................................................... 70
Cellulaire oorsprong van het product .......................................................................................................... 70
Expressie in Pichia pastoris .......................................................................................................................... 71
Stabiele integratie van expressieconstructen in het genoom .................................................................... 71
Alcohol oxidase promotor en het Alfa mating signaal............................................................................... 72
Belang codon optimalisatie ..................................................................................................................... 73
Kloonselectie .............................................................................................................................................. 74
Fermentatie ................................................................................................................................................ 74
Multicistronische boodschappers .................................................................................................................... 75
IRES – mechanisme ..................................................................................................................................... 75
2A peptide .................................................................................................................................................. 76
4
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 matissedecaluwe. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $8.65. You're not tied to anything after your purchase.
