Inhoud
1 DNA-klonering...........................................................................................5
1.1 PCR.....................................................................................................5
1.1.1 DNA-polymerase...........................................................................5
1.1.2 Template concentratie..................................................................6
1.1.3 Analyse van PCR producten..........................................................7
1.1.4 Aspecifieke amplicons..................................................................9
1.1.5 Verwijderen van het template DNA..............................................9
1.2 Isolatie van genomisch DNA.............................................................10
1.2.1 Cellyse........................................................................................10
1.2.2 DNA zuivering.............................................................................12
1.3 Synthetische DNA aanmaak..............................................................14
1.3.1 Ontwerp DNA sequentie.............................................................14
1.3.2 Chemische DNA synthese...........................................................14
1.3.3 Enzymatische DNA synthese......................................................15
1.3.4 Vertrekken uit RNA.....................................................................15
1.4 DNA inbrengen in een plasmide.......................................................16
1.4.1 Restrictie-ligation........................................................................16
1.4.2 Gibson assembly.........................................................................16
1.4.3 Golden gate assembly................................................................18
1.4.4 Circular Polymerase Extension Cloning (=CPEC)........................18
1.4.5 SLIC.............................................................................................18
1.5 Transformatie van cellen..................................................................19
1.5.1 Electrocompetente cellen...........................................................19
1.5.2 Chemisch competente cellen......................................................19
1.6 Selectie van getransformeerde cellen..............................................21
1.6.1 Positieve selectiemerkers...........................................................21
1.6.2 Negatieve selectiemerkers.........................................................22
1.7 Vermenigvuldiging van het plasmide...............................................22
1.8 Isolatie van het plasmide-DNA..........................................................23
1.8.1 Mini-, midi-, maxiprep.................................................................23
1.8.2 Bepaling van DNA-concentratie van plasmide............................23
, 1.8.3 Verificatie van het insert.............................................................23
2 Gerichte genoommodificaties in prokaryoten en eukaryoten.................24
2.1 Biologische functie van CRISPR-Cas..................................................24
2.1.1 Adaptatie en sensitisatie............................................................24
2.1.2 Transcriptie en verwerking.........................................................25
2.1.3 Interferentie of effector fase.......................................................26
2.2 Cas-varianten en hun specifieke toepassingen.................................26
2.2.1 Cas9............................................................................................26
2.2.2 Cas12..........................................................................................29
2.2.3 Cas13..........................................................................................29
2.3 Aanpassingen om CRIPSR-CAS te gebruiken in eukaryote cellen.....29
2.3.1 Delivery methoden.....................................................................30
2.4 Minimaliseren van off-target effecten...............................................30
2.4.1 Optimaliseer gRNA ontwerp........................................................30
2.4.2 Gebruik hoog specifieke Cas9 varianten....................................31
2.4.3 Verkort de gDNA lengte..............................................................31
2.4.4 Dubbele nickase strategie..........................................................31
2.4.5 FokI-dCas9..................................................................................31
2.4.6 Onderzoek off-target knipplaatsen experimenteel.....................32
2.5 Base editing......................................................................................32
2.5.1 Cytosine base editors (CBE).......................................................32
2.5.2 Adenine base editors..................................................................33
2.6 Prime-editing....................................................................................33
3 Omics technologieën...............................................................................34
3.1 Genomics..........................................................................................34
3.1.1 Next generation sequencing.......................................................34
3.2 Transcriptomics................................................................................37
3.2.1 Technieken.................................................................................37
3.2.2 RNA sequencing..........................................................................38
3.3 Proteomics........................................................................................40
3.3.1 Scheidingstechnieken.................................................................40
3.3.2 Ionisatietechnieken.....................................................................41
3.3.3 Detectietechnieken.....................................................................41
, 3.4 Metabolomics....................................................................................42
3.5 Epigenetics.......................................................................................43
3.5.1 DNA-methylering........................................................................43
3.5.2 Histon modificaties.....................................................................44
3.5.3 Niet coderend RNA.....................................................................45
4 Kanker.....................................................................................................46
4.1 6 hoofdkenmerken van kanker.........................................................46
4.1.1 Aanhoudende proliferatieve signalen.........................................46
4.1.2 Ontsnappen aan groei inhibitie...................................................46
4.1.3 Verwerven van capaciteit tot oneindige celdeling......................47
4.1.4 Resistentie aan actieve celdood-processen................................47
4.1.5 Neoangiogenese: vorming van nieuwe bloedvaten....................48
4.1.6 Invasie en metastase..................................................................48
4.2 Genetische basis van kanker............................................................48
4.2.1 Proto-oncogenen.........................................................................48
4.2.2 Tumor-suppressorgenen.............................................................48
4.2.3 Celdoodgenen.............................................................................48
4.2.4 Caretaker genen.........................................................................49
4.2.5 Gatekeeper genen......................................................................49
4.3 Soorten tumoren...............................................................................49
4.4 Het ‘multi-hit’ model van kanker......................................................49
4.5 Recente begrippen omtrent kanker..................................................50
4.5.1 Ontregeling van het cellulaire energiemetabolisme...................50
4.5.2 Tumor bevorderende inflammatie..............................................50
4.5.3 Ontwijken van immunologische vernieling.................................50
5 Proteïne-Proteïne interacties...................................................................52
5.1 Faagdisplay.......................................................................................52
5.2 Yeast diplay......................................................................................52
5.3 Mammalian display...........................................................................52
5.4 Bacterial display...............................................................................53
5.5 Ribosoom display..............................................................................53
5.6 Yeast-two-hydrid (Y2H) system.........................................................55
5.7 Co-immunoprecipitatie (co-IP)..........................................................55
, 5.8 Pull-down assay................................................................................56
5.9 Protein microarray............................................................................56
5.10 Fluorescence resonance energy transfer (FRET)............................56
5.11 Proximity ligation assay..................................................................56
1 DNA-klonering...........................................................................................5
1.1 PCR.....................................................................................................5
1.1.1 DNA-polymerase...........................................................................5
1.1.2 Template concentratie..................................................................6
1.1.3 Analyse van PCR producten..........................................................7
1.1.4 Aspecifieke amplicons..................................................................9
1.1.5 Verwijderen van het template DNA..............................................9
1.2 Isolatie van genomisch DNA.............................................................10
1.2.1 Cellyse........................................................................................10
1.2.2 DNA zuivering.............................................................................12
1.3 Synthetische DNA aanmaak..............................................................14
1.3.1 Ontwerp DNA sequentie.............................................................14
1.3.2 Chemische DNA synthese...........................................................14
1.3.3 Enzymatische DNA synthese......................................................15
1.3.4 Vertrekken uit RNA.....................................................................15
1.4 DNA inbrengen in een plasmide.......................................................16
1.4.1 Restrictie-ligation........................................................................16
1.4.2 Gibson assembly.........................................................................16
1.4.3 Golden gate assembly................................................................18
1.4.4 Circular Polymerase Extension Cloning (=CPEC)........................18
1.4.5 SLIC.............................................................................................18
1.5 Transformatie van cellen..................................................................19
1.5.1 Electrocompetente cellen...........................................................19
1.5.2 Chemisch competente cellen......................................................19
1.6 Selectie van getransformeerde cellen..............................................21
1.6.1 Positieve selectiemerkers...........................................................21
1.6.2 Negatieve selectiemerkers.........................................................22
1.7 Vermenigvuldiging van het plasmide...............................................22
1.8 Isolatie van het plasmide-DNA..........................................................23
1.8.1 Mini-, midi-, maxiprep.................................................................23
1.8.2 Bepaling van DNA-concentratie van plasmide............................23
, 1.8.3 Verificatie van het insert.............................................................23
2 Gerichte genoommodificaties in prokaryoten en eukaryoten.................24
2.1 Biologische functie van CRISPR-Cas..................................................24
2.1.1 Adaptatie en sensitisatie............................................................24
2.1.2 Transcriptie en verwerking.........................................................25
2.1.3 Interferentie of effector fase.......................................................26
2.2 Cas-varianten en hun specifieke toepassingen.................................26
2.2.1 Cas9............................................................................................26
2.2.2 Cas12..........................................................................................29
2.2.3 Cas13..........................................................................................29
2.3 Aanpassingen om CRIPSR-CAS te gebruiken in eukaryote cellen.....29
2.3.1 Delivery methoden.....................................................................30
2.4 Minimaliseren van off-target effecten...............................................30
2.4.1 Optimaliseer gRNA ontwerp........................................................30
2.4.2 Gebruik hoog specifieke Cas9 varianten....................................31
2.4.3 Verkort de gDNA lengte..............................................................31
2.4.4 Dubbele nickase strategie..........................................................31
2.4.5 FokI-dCas9..................................................................................31
2.4.6 Onderzoek off-target knipplaatsen experimenteel.....................32
2.5 Base editing......................................................................................32
2.5.1 Cytosine base editors (CBE).......................................................32
2.5.2 Adenine base editors..................................................................33
2.6 Prime-editing....................................................................................33
3 Omics technologieën...............................................................................34
3.1 Genomics..........................................................................................34
3.1.1 Next generation sequencing.......................................................34
3.2 Transcriptomics................................................................................37
3.2.1 Technieken.................................................................................37
3.2.2 RNA sequencing..........................................................................38
3.3 Proteomics........................................................................................40
3.3.1 Scheidingstechnieken.................................................................40
3.3.2 Ionisatietechnieken.....................................................................41
3.3.3 Detectietechnieken.....................................................................41
, 3.4 Metabolomics....................................................................................42
3.5 Epigenetics.......................................................................................43
3.5.1 DNA-methylering........................................................................43
3.5.2 Histon modificaties.....................................................................44
3.5.3 Niet coderend RNA.....................................................................45
4 Kanker.....................................................................................................46
4.1 6 hoofdkenmerken van kanker.........................................................46
4.1.1 Aanhoudende proliferatieve signalen.........................................46
4.1.2 Ontsnappen aan groei inhibitie...................................................46
4.1.3 Verwerven van capaciteit tot oneindige celdeling......................47
4.1.4 Resistentie aan actieve celdood-processen................................47
4.1.5 Neoangiogenese: vorming van nieuwe bloedvaten....................48
4.1.6 Invasie en metastase..................................................................48
4.2 Genetische basis van kanker............................................................48
4.2.1 Proto-oncogenen.........................................................................48
4.2.2 Tumor-suppressorgenen.............................................................48
4.2.3 Celdoodgenen.............................................................................48
4.2.4 Caretaker genen.........................................................................49
4.2.5 Gatekeeper genen......................................................................49
4.3 Soorten tumoren...............................................................................49
4.4 Het ‘multi-hit’ model van kanker......................................................49
4.5 Recente begrippen omtrent kanker..................................................50
4.5.1 Ontregeling van het cellulaire energiemetabolisme...................50
4.5.2 Tumor bevorderende inflammatie..............................................50
4.5.3 Ontwijken van immunologische vernieling.................................50
5 Proteïne-Proteïne interacties...................................................................52
5.1 Faagdisplay.......................................................................................52
5.2 Yeast diplay......................................................................................52
5.3 Mammalian display...........................................................................52
5.4 Bacterial display...............................................................................53
5.5 Ribosoom display..............................................................................53
5.6 Yeast-two-hydrid (Y2H) system.........................................................55
5.7 Co-immunoprecipitatie (co-IP)..........................................................55
, 5.8 Pull-down assay................................................................................56
5.9 Protein microarray............................................................................56
5.10 Fluorescence resonance energy transfer (FRET)............................56
5.11 Proximity ligation assay..................................................................56
