Samenvatting Farmaceutische biotechnologie
Bach 1 – SEM 2
Inhoudsopgave
Hoofdstuk 1: introductie ..........................................................................................................6
Wat is nu juist biotechnologie? .........................................................................................6
Wat is het belang van biotechnologie? ..............................................................................6
Oorsprong van de 1ste biologische geneesmiddelen ...........................................................7
Biotechnologie tijdens de opleiding ...................................................................................8
Centrale dogma : replicatie – transcriptie – translatie ........................................................9
Hoofdstuk 2: manipuleren van DNA ....................................................................................... 10
2.1 Plasmiden.................................................................................................................... 10
2.2 Restrictie enzymen ....................................................................................................... 11
DNA knippen en plakken................................................................................................. 13
Oefening DNA kloneren .................................................................................................. 13
2.3: DNA scheidingstechnieken .......................................................................................... 14
Agarose gelelektroforese ................................................................................................ 14
2.4: transformatie .............................................................................................................. 16
Chemische transformatie ............................................................................................... 16
Elektroporatie ................................................................................................................ 16
2.5: DNA zuiveren .............................................................................................................. 17
Hoe zuiveren we DNA/RNA ? ........................................................................................... 17
Isolatie van chromosomaal DNA.................................................................................... 17
Isolatie van plasmide DNA ............................................................................................. 18
Alternatieve methode: paramagnetische partikels ........................................................... 18
2.6: bronnen van DNA (Southern Blot & PCR) ...................................................................... 19
Bron van DNA: genomisch .............................................................................................. 19
Bron van DNA: cDNA ...................................................................................................... 21
2.7: sequentiebepaling....................................................................................................... 25
H3: Productie proces recombinante eiwitten: expressie ......................................................... 28
3.1: introductie: productie systemen .................................................................................. 28
3.2: posttranslationele modificaties ................................................................................... 28
Glycosylatie (N-glycosylatie & O-glycosylatie) ................................................................. 29
Zwavelbruggen ............................................................................................................... 29
Proteolyse...................................................................................................................... 30
1
, Post productie & zuivering .............................................................................................. 30
3.3: plasmiden voor expressie ............................................................................................ 31
Regelbare promotor ....................................................................................................... 31
Ribosome binding site: RNA → eiwit ............................................................................... 33
Coderende sequentie ..................................................................................................... 33
Terminator sequentie ..................................................................................................... 33
3.4: Transformatie / transfectie .......................................................................................... 34
3.5: eiwitproductie: gastheren ............................................................................................ 35
Bacterieel ...................................................................................................................... 35
Gisten (eukaryoot) .......................................................................................................... 37
Transgene dieren ............................................................................................................ 39
Planten .......................................................................................................................... 40
Zoogdiercellen ............................................................................................................... 40
3.6: eiwitproductie: technieken .......................................................................................... 41
H4: Opzuiveren van recombinante eiwitten ............................................................................ 44
4.1: introductie .................................................................................................................. 44
4.2: staalvoorbereiding ...................................................................................................... 44
Centrifugatie .................................................................................................................. 44
Dialyse........................................................................................................................... 45
Cellen openbreken ......................................................................................................... 45
Filtratie .......................................................................................................................... 46
Precipitatie .................................................................................................................... 47
4.3: chromatografie ........................................................................................................... 47
Ionenuitwisselingschromatografie .................................................................................. 48
Affiniteitschromatografie ................................................................................................ 48
Hydrofobe interactiechromatografie ............................................................................... 50
Gelfiltratie ...................................................................................................................... 51
Hoofdstuk 5: Eiwit karakterisatie ............................................................................................ 54
5.1: introductie antilichamen ............................................................................................. 54
5.2: concentratie bepaling.................................................................................................. 57
UV-spectrofotometrie (directe methode)......................................................................... 57
Indirecte methode: Bradford ........................................................................................... 58
Absolute kwantificatie .................................................................................................... 60
5.3: scheiding op basis van grootte: SDS-PAGE ................................................................... 61
5.4: Western blot ............................................................................................................... 63
5.5: scheiding op basis van lading....................................................................................... 63
2
, Native Page .................................................................................................................... 63
Isoelektrisch focussing ................................................................................................... 64
5.6: 2D-elektroforese ......................................................................................................... 64
5.7: ELISA .......................................................................................................................... 65
Directe ELISA ................................................................................................................. 66
Indirecte ELISA ............................................................................................................... 66
Sandwich ELISA ............................................................................................................. 67
Competitie ELISA ........................................................................................................... 67
ELISA - oefening ............................................................................................................. 68
5.8: Fluorescentie gebaseerde technieken .......................................................................... 69
Microscopie ................................................................................................................... 70
Flow cytometrie ............................................................................................................. 71
Quenching ..................................................................................................................... 72
FRET .............................................................................................................................. 74
Fluorescente eiwitten ..................................................................................................... 76
5.9: bioluminescentie ........................................................................................................ 77
5.10: reporter assays ......................................................................................................... 78
5.11: surface plasmon resonance ...................................................................................... 79
Hoofdstuk 6: enzymen gebruikt bij recombinante DNA technologie ......................................... 82
6.1: Ligasen ....................................................................................................................... 82
6.2: Fosfatasen .................................................................................................................. 84
6.3: kinasen ....................................................................................................................... 84
6.4: Methyltransferasen ..................................................................................................... 85
6.5: polymerasen ............................................................................................................... 86
6.6: nucleasen ................................................................................................................... 88
6.7: recombinasen ............................................................................................................. 89
Hoofdstuk 7: PCR-technieken ................................................................................................ 92
7.1: conventionele PCR ...................................................................................................... 92
7.2: Site directed mutagenese – basen verwijderen, veranderen of toevoegen ...................... 92
Conventionele PCR met mutagene primers ..................................................................... 92
Conventionele PCR met verkorte primers (deleties) ......................................................... 93
Conventionele PCR met verlengde primers (inserties)...................................................... 93
Primer extensie mutagenese voor inserties .................................................................... 96
Primer extensie mutagenese voor deleties ..................................................................... 97
7.3: Nested PCR – verhoogt de specificiteit ......................................................................... 97
7.4: hot start PCR – verhoogt de specificiteit ....................................................................... 98
3
, 7.5: touch down PCR – verhoogt de specificiteit .................................................................. 98
7.6: Error prone PCR – willekeurige mutaties ..................................................................... 100
7.7: Kolonie PCR – PCR op bacteriële kolonies .................................................................. 100
7.8: qPCR – kwantitatieve PCR ......................................................................................... 102
7.9: digital PCR ................................................................................................................ 106
Hoofdstuk 8: DNA/RNA analyse & next generation sequencing .............................................. 108
8.1: DNA-eiwit interactie .................................................................................................. 108
Gel mobility shift essay ................................................................................................. 108
DNAse I foot printing .................................................................................................... 108
ChIP-Seq ..................................................................................................................... 109
8.2: DNA/RNA: identificeren van start/stop regio’s ............................................................ 110
Nuclease S1 mapping ................................................................................................... 110
Primer extensie ............................................................................................................ 111
8.3: bepalen van DNA/RNA samenstelling (zonder sequeneren) ........................................ 112
PCR blockers ter bepaling van SNPs (single nucleotide polymorphism) .......................... 112
RAPD – randomly amplified polymorphic DNA ............................................................... 113
RFLP – restriction fragment length polymorphism .......................................................... 113
AFLP – amplified fragment length polymorpism ............................................................. 114
CAPS – cleaved amplified polymorphic sequence.......................................................... 115
PCR op VNTR (Variable Number of Tandem Repeats) regio’s .......................................... 116
LAMP – loop-mediated isothermal AMPlification............................................................ 116
Microarrays .................................................................................................................. 118
8.3: Next generation sequencing ...................................................................................... 119
Second generation sequencing ..................................................................................... 120
Third generation sequencing ......................................................................................... 127
Dataverwerking & toepassingen .................................................................................... 130
Hoofdstuk 9: display technieken .......................................................................................... 132
9.1: inleiding .................................................................................................................... 132
9.2: faagdisplay ............................................................................................................... 133
9.3: gistdisplay ................................................................................................................ 138
Hoofdstuk 10: genoommanipulatie ...................................................................................... 142
10.2: inleiding .................................................................................................................. 142
10.2: homologe recombinatie........................................................................................... 142
10.3: CRISPR/Cas ............................................................................................................ 146
NHEJ - Non-homologous end joining ............................................................................. 148
Homology directed repair ............................................................................................. 148
4
Bach 1 – SEM 2
Inhoudsopgave
Hoofdstuk 1: introductie ..........................................................................................................6
Wat is nu juist biotechnologie? .........................................................................................6
Wat is het belang van biotechnologie? ..............................................................................6
Oorsprong van de 1ste biologische geneesmiddelen ...........................................................7
Biotechnologie tijdens de opleiding ...................................................................................8
Centrale dogma : replicatie – transcriptie – translatie ........................................................9
Hoofdstuk 2: manipuleren van DNA ....................................................................................... 10
2.1 Plasmiden.................................................................................................................... 10
2.2 Restrictie enzymen ....................................................................................................... 11
DNA knippen en plakken................................................................................................. 13
Oefening DNA kloneren .................................................................................................. 13
2.3: DNA scheidingstechnieken .......................................................................................... 14
Agarose gelelektroforese ................................................................................................ 14
2.4: transformatie .............................................................................................................. 16
Chemische transformatie ............................................................................................... 16
Elektroporatie ................................................................................................................ 16
2.5: DNA zuiveren .............................................................................................................. 17
Hoe zuiveren we DNA/RNA ? ........................................................................................... 17
Isolatie van chromosomaal DNA.................................................................................... 17
Isolatie van plasmide DNA ............................................................................................. 18
Alternatieve methode: paramagnetische partikels ........................................................... 18
2.6: bronnen van DNA (Southern Blot & PCR) ...................................................................... 19
Bron van DNA: genomisch .............................................................................................. 19
Bron van DNA: cDNA ...................................................................................................... 21
2.7: sequentiebepaling....................................................................................................... 25
H3: Productie proces recombinante eiwitten: expressie ......................................................... 28
3.1: introductie: productie systemen .................................................................................. 28
3.2: posttranslationele modificaties ................................................................................... 28
Glycosylatie (N-glycosylatie & O-glycosylatie) ................................................................. 29
Zwavelbruggen ............................................................................................................... 29
Proteolyse...................................................................................................................... 30
1
, Post productie & zuivering .............................................................................................. 30
3.3: plasmiden voor expressie ............................................................................................ 31
Regelbare promotor ....................................................................................................... 31
Ribosome binding site: RNA → eiwit ............................................................................... 33
Coderende sequentie ..................................................................................................... 33
Terminator sequentie ..................................................................................................... 33
3.4: Transformatie / transfectie .......................................................................................... 34
3.5: eiwitproductie: gastheren ............................................................................................ 35
Bacterieel ...................................................................................................................... 35
Gisten (eukaryoot) .......................................................................................................... 37
Transgene dieren ............................................................................................................ 39
Planten .......................................................................................................................... 40
Zoogdiercellen ............................................................................................................... 40
3.6: eiwitproductie: technieken .......................................................................................... 41
H4: Opzuiveren van recombinante eiwitten ............................................................................ 44
4.1: introductie .................................................................................................................. 44
4.2: staalvoorbereiding ...................................................................................................... 44
Centrifugatie .................................................................................................................. 44
Dialyse........................................................................................................................... 45
Cellen openbreken ......................................................................................................... 45
Filtratie .......................................................................................................................... 46
Precipitatie .................................................................................................................... 47
4.3: chromatografie ........................................................................................................... 47
Ionenuitwisselingschromatografie .................................................................................. 48
Affiniteitschromatografie ................................................................................................ 48
Hydrofobe interactiechromatografie ............................................................................... 50
Gelfiltratie ...................................................................................................................... 51
Hoofdstuk 5: Eiwit karakterisatie ............................................................................................ 54
5.1: introductie antilichamen ............................................................................................. 54
5.2: concentratie bepaling.................................................................................................. 57
UV-spectrofotometrie (directe methode)......................................................................... 57
Indirecte methode: Bradford ........................................................................................... 58
Absolute kwantificatie .................................................................................................... 60
5.3: scheiding op basis van grootte: SDS-PAGE ................................................................... 61
5.4: Western blot ............................................................................................................... 63
5.5: scheiding op basis van lading....................................................................................... 63
2
, Native Page .................................................................................................................... 63
Isoelektrisch focussing ................................................................................................... 64
5.6: 2D-elektroforese ......................................................................................................... 64
5.7: ELISA .......................................................................................................................... 65
Directe ELISA ................................................................................................................. 66
Indirecte ELISA ............................................................................................................... 66
Sandwich ELISA ............................................................................................................. 67
Competitie ELISA ........................................................................................................... 67
ELISA - oefening ............................................................................................................. 68
5.8: Fluorescentie gebaseerde technieken .......................................................................... 69
Microscopie ................................................................................................................... 70
Flow cytometrie ............................................................................................................. 71
Quenching ..................................................................................................................... 72
FRET .............................................................................................................................. 74
Fluorescente eiwitten ..................................................................................................... 76
5.9: bioluminescentie ........................................................................................................ 77
5.10: reporter assays ......................................................................................................... 78
5.11: surface plasmon resonance ...................................................................................... 79
Hoofdstuk 6: enzymen gebruikt bij recombinante DNA technologie ......................................... 82
6.1: Ligasen ....................................................................................................................... 82
6.2: Fosfatasen .................................................................................................................. 84
6.3: kinasen ....................................................................................................................... 84
6.4: Methyltransferasen ..................................................................................................... 85
6.5: polymerasen ............................................................................................................... 86
6.6: nucleasen ................................................................................................................... 88
6.7: recombinasen ............................................................................................................. 89
Hoofdstuk 7: PCR-technieken ................................................................................................ 92
7.1: conventionele PCR ...................................................................................................... 92
7.2: Site directed mutagenese – basen verwijderen, veranderen of toevoegen ...................... 92
Conventionele PCR met mutagene primers ..................................................................... 92
Conventionele PCR met verkorte primers (deleties) ......................................................... 93
Conventionele PCR met verlengde primers (inserties)...................................................... 93
Primer extensie mutagenese voor inserties .................................................................... 96
Primer extensie mutagenese voor deleties ..................................................................... 97
7.3: Nested PCR – verhoogt de specificiteit ......................................................................... 97
7.4: hot start PCR – verhoogt de specificiteit ....................................................................... 98
3
, 7.5: touch down PCR – verhoogt de specificiteit .................................................................. 98
7.6: Error prone PCR – willekeurige mutaties ..................................................................... 100
7.7: Kolonie PCR – PCR op bacteriële kolonies .................................................................. 100
7.8: qPCR – kwantitatieve PCR ......................................................................................... 102
7.9: digital PCR ................................................................................................................ 106
Hoofdstuk 8: DNA/RNA analyse & next generation sequencing .............................................. 108
8.1: DNA-eiwit interactie .................................................................................................. 108
Gel mobility shift essay ................................................................................................. 108
DNAse I foot printing .................................................................................................... 108
ChIP-Seq ..................................................................................................................... 109
8.2: DNA/RNA: identificeren van start/stop regio’s ............................................................ 110
Nuclease S1 mapping ................................................................................................... 110
Primer extensie ............................................................................................................ 111
8.3: bepalen van DNA/RNA samenstelling (zonder sequeneren) ........................................ 112
PCR blockers ter bepaling van SNPs (single nucleotide polymorphism) .......................... 112
RAPD – randomly amplified polymorphic DNA ............................................................... 113
RFLP – restriction fragment length polymorphism .......................................................... 113
AFLP – amplified fragment length polymorpism ............................................................. 114
CAPS – cleaved amplified polymorphic sequence.......................................................... 115
PCR op VNTR (Variable Number of Tandem Repeats) regio’s .......................................... 116
LAMP – loop-mediated isothermal AMPlification............................................................ 116
Microarrays .................................................................................................................. 118
8.3: Next generation sequencing ...................................................................................... 119
Second generation sequencing ..................................................................................... 120
Third generation sequencing ......................................................................................... 127
Dataverwerking & toepassingen .................................................................................... 130
Hoofdstuk 9: display technieken .......................................................................................... 132
9.1: inleiding .................................................................................................................... 132
9.2: faagdisplay ............................................................................................................... 133
9.3: gistdisplay ................................................................................................................ 138
Hoofdstuk 10: genoommanipulatie ...................................................................................... 142
10.2: inleiding .................................................................................................................. 142
10.2: homologe recombinatie........................................................................................... 142
10.3: CRISPR/Cas ............................................................................................................ 146
NHEJ - Non-homologous end joining ............................................................................. 148
Homology directed repair ............................................................................................. 148
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