Inhoudsopgave
METHODEN IN BIOMEDISCH ONDERZOEK 3 ........................................... 5
3.1 INLEIDING ............................................................................................ 5
3.2 DNA: DRAGER GENETISCHE INFORMATIE...................................................... 6
3.2.1.1 1STE GENERATIE SEQUENTIENEPALING ....................................................... 9
3.3.1.2 2DE GENERATIE SEQUENTIEBEPALING .................................................... 11
3.2.1.3 3DE GENERATIE SEQUENTIEBEPALING .................................................... 30
3.2.1.4 EPIGENETICA SEQUENTIEBEPALING ...................................................... 32
3.2.1.5 Single-cell sequen3ebepaling ........................................................ 34
3.2.2 DNA GENOTYPEREN ............................................................................... 36
3.2.2.1 Short tandem repeat genotypering ............................................... 38
3.2.2.2 Taqman genotypering.................................................................... 40
3.2.2.3 Microarrays (voor bepaling DNA sequen3e varia3e) .................... 41
3.2.3 DNA HYDRIDIZEREN ............................................................................... 46
3.2.3.1 FISH ............................................................................................... 47
3.2.3.2 CGH ............................................................................................... 47
3.3 RNA ................................................................................................ 47
3.3.1 RNA MICROARRAYS ............................................................................... 49
3.3.2 RNA SEQUENCING RNA-SEQ (BULK) ......................................................... 54
3.3.2.1 short read cDNA sequencing ......................................................... 54
3.3.2.2 long read cDNA sequencing .......................................................... 57
3.3.3 SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ) ............................................. 57
3.3.4. SPATIAL TRANSCRIPTOMICS ..................................................................... 63
3.4 EIWITTEN ........................................................................................... 67
3.4.1 EIWIT SEQUENTIBEPALING ........................................................................ 68
3.4.1.1 Edman degrada3e ......................................................................... 68
3.4.1.2 eiwit sequen3ebepaling via massa spectrometrie ........................ 68
3.4.2.1 Bulk analyse: proteomics op eiwitextracten.................................. 70
3.4.2.2 Single cell proteomics.................................................................... 76
1
,3.4.2.3 Spa3al proteomics ......................................................................... 79
3.4.3 3D STRUCTUURBEP ALING .................................................................... 81
3.4.3.1 X-straaldiffrac3e en kristallografie................................................. 82
3.4.3.2 NMR spectroscopie ....................................................................... 89
3.4.3.3 Cryo-elektronenmicroscopie (Cryo-EM) ........................................ 90
3.4.4 EIWIT- EIWIT EN EIWT-LIGAND INTERACTIES ................................................. 90
3.4.4.1 Yeast-2-hybrid en mammalian 2-hybrid ........................................ 91
3.4.4.2 Affinity pull-down .......................................................................... 93
3.4.4.3 Co-immunoprecipita3e (co-IP) ...................................................... 94
3.4.4.4 FRET en BRET ................................................................................. 95
3.4.4.5 Bindingsarrays ............................................................................... 96
3.4.4.6 Surface plasmon resonance .......................................................... 97
3.4.4.7 phage display ................................................................................. 97
3.4.4.8 Proximity liga3on assay ................................................................. 98
3.4.4.9 Ligand – receptor bindingstudies .................................................. 99
3.5 ANALYSE VAN METABOLIETEN EN LIPIDEN .................................................. 100
3.5.1 SPECIFIEKE TESTEN VAN INDIVIDUELE METABOLIETEN .................................... 101
3.5.2 METABOLOMICS ................................................................................... 102
3.5.3 METABOLE FLUX ANALYSE ....................................................................... 105
3.5.4 ANALYSE VAN LIPIDEN ............................................................................ 106
3.6 ANALYSE VAN GENTRANSCRIPTIE REGULATIE .............................................. 121
3.6.1 NASCENT RNA ANALYSES ...................................................................... 121
3.6.1.1 Run-on assays .............................................................................. 121
3.6.1.2 Andere nascent RNA analyse methoden ..................................... 123
3.6.1.3 Imaging-gebaseerde nascent RNA analyse methoden ................ 126
3.6.2 PROMOTOR REPORTER STUDIES............................................................... 126
3.6.3 ANALYSE VAN DNA-PROTEÏNE INTERACTIES ............................................... 127
3.6.3.1 DNAse footprin3ng ...................................................................... 127
3.6.3.2 EMSA: electrophore3c mobility shi` assay ................................. 128
3.6.3.3 Open chroma3ne assays ............................................................. 129
3.6.3.4 ChIP, ChIP-chip en ChIP-seq ......................................................... 132
3.7 DATA-ANALYSE EN DATABANKEN ............................................................ 133
2
,3.7.1.1 Literatuur ..................................................................................... 134
3.7.1.2 Data Respositora ......................................................................... 135
3.7.1.3 Data placormen .......................................................................... 135
3.7.2 DATA ANALYSE ..................................................................................... 137
3.7.2.1 Annota3e van genlijsten .............................................................. 137
3.7.2.2 interac3enetwerken .................................................................... 138
3.7.2.3 structuuranalyse van biomoleculen ............................................ 138
3.8 MODULATIE VAN GENEXPRESSIE ............................................................. 140
3.8.1 ONDERDRUKKING VAN GENEXPRESSIE VIA ANTISENSE TECHNOLOGIE ................ 140
3.8.1.1 An3sense oligonucelo3de ........................................................... 140
3.8.1.2 RNA interferen3e (RNAi) ............................................................. 141
3.8.2 GENDISRUPTIE, MUTAGENESE EN EDITING .................................................. 146
3.8.2.1 transposons ................................................................................. 146
3.8.2.4 CRISPR genoommodifica3e ......................................................... 151
3.8.3 ECTOPISCHE EXPRESSIE EN OVEREXPRESSIE ................................................ 161
3.8.3.1 directe mRNA injec3e.................................................................. 161
3.8.3.2 DNA vectoren .............................................................................. 162
3.8.4 GERICHTE PROTEÏNE DEGRADATIE ............................................................. 166
3.8.4.1 Condi3onele degrons .................................................................. 166
... 168
3
, ... 168
3.8.4.2 Bifunc3onele moleculen ............................................................. 168
3.8.4.3 TRIM away ................................................................................... 169
3.8.5 METHODEN VAN GENTRANSFER ............................................................... 169
3.8.5.1 transfec3e ................................................................................... 169
3.8.5.2 Transduc3e .................................................................................. 172
4
METHODEN IN BIOMEDISCH ONDERZOEK 3 ........................................... 5
3.1 INLEIDING ............................................................................................ 5
3.2 DNA: DRAGER GENETISCHE INFORMATIE...................................................... 6
3.2.1.1 1STE GENERATIE SEQUENTIENEPALING ....................................................... 9
3.3.1.2 2DE GENERATIE SEQUENTIEBEPALING .................................................... 11
3.2.1.3 3DE GENERATIE SEQUENTIEBEPALING .................................................... 30
3.2.1.4 EPIGENETICA SEQUENTIEBEPALING ...................................................... 32
3.2.1.5 Single-cell sequen3ebepaling ........................................................ 34
3.2.2 DNA GENOTYPEREN ............................................................................... 36
3.2.2.1 Short tandem repeat genotypering ............................................... 38
3.2.2.2 Taqman genotypering.................................................................... 40
3.2.2.3 Microarrays (voor bepaling DNA sequen3e varia3e) .................... 41
3.2.3 DNA HYDRIDIZEREN ............................................................................... 46
3.2.3.1 FISH ............................................................................................... 47
3.2.3.2 CGH ............................................................................................... 47
3.3 RNA ................................................................................................ 47
3.3.1 RNA MICROARRAYS ............................................................................... 49
3.3.2 RNA SEQUENCING RNA-SEQ (BULK) ......................................................... 54
3.3.2.1 short read cDNA sequencing ......................................................... 54
3.3.2.2 long read cDNA sequencing .......................................................... 57
3.3.3 SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ) ............................................. 57
3.3.4. SPATIAL TRANSCRIPTOMICS ..................................................................... 63
3.4 EIWITTEN ........................................................................................... 67
3.4.1 EIWIT SEQUENTIBEPALING ........................................................................ 68
3.4.1.1 Edman degrada3e ......................................................................... 68
3.4.1.2 eiwit sequen3ebepaling via massa spectrometrie ........................ 68
3.4.2.1 Bulk analyse: proteomics op eiwitextracten.................................. 70
3.4.2.2 Single cell proteomics.................................................................... 76
1
,3.4.2.3 Spa3al proteomics ......................................................................... 79
3.4.3 3D STRUCTUURBEP ALING .................................................................... 81
3.4.3.1 X-straaldiffrac3e en kristallografie................................................. 82
3.4.3.2 NMR spectroscopie ....................................................................... 89
3.4.3.3 Cryo-elektronenmicroscopie (Cryo-EM) ........................................ 90
3.4.4 EIWIT- EIWIT EN EIWT-LIGAND INTERACTIES ................................................. 90
3.4.4.1 Yeast-2-hybrid en mammalian 2-hybrid ........................................ 91
3.4.4.2 Affinity pull-down .......................................................................... 93
3.4.4.3 Co-immunoprecipita3e (co-IP) ...................................................... 94
3.4.4.4 FRET en BRET ................................................................................. 95
3.4.4.5 Bindingsarrays ............................................................................... 96
3.4.4.6 Surface plasmon resonance .......................................................... 97
3.4.4.7 phage display ................................................................................. 97
3.4.4.8 Proximity liga3on assay ................................................................. 98
3.4.4.9 Ligand – receptor bindingstudies .................................................. 99
3.5 ANALYSE VAN METABOLIETEN EN LIPIDEN .................................................. 100
3.5.1 SPECIFIEKE TESTEN VAN INDIVIDUELE METABOLIETEN .................................... 101
3.5.2 METABOLOMICS ................................................................................... 102
3.5.3 METABOLE FLUX ANALYSE ....................................................................... 105
3.5.4 ANALYSE VAN LIPIDEN ............................................................................ 106
3.6 ANALYSE VAN GENTRANSCRIPTIE REGULATIE .............................................. 121
3.6.1 NASCENT RNA ANALYSES ...................................................................... 121
3.6.1.1 Run-on assays .............................................................................. 121
3.6.1.2 Andere nascent RNA analyse methoden ..................................... 123
3.6.1.3 Imaging-gebaseerde nascent RNA analyse methoden ................ 126
3.6.2 PROMOTOR REPORTER STUDIES............................................................... 126
3.6.3 ANALYSE VAN DNA-PROTEÏNE INTERACTIES ............................................... 127
3.6.3.1 DNAse footprin3ng ...................................................................... 127
3.6.3.2 EMSA: electrophore3c mobility shi` assay ................................. 128
3.6.3.3 Open chroma3ne assays ............................................................. 129
3.6.3.4 ChIP, ChIP-chip en ChIP-seq ......................................................... 132
3.7 DATA-ANALYSE EN DATABANKEN ............................................................ 133
2
,3.7.1.1 Literatuur ..................................................................................... 134
3.7.1.2 Data Respositora ......................................................................... 135
3.7.1.3 Data placormen .......................................................................... 135
3.7.2 DATA ANALYSE ..................................................................................... 137
3.7.2.1 Annota3e van genlijsten .............................................................. 137
3.7.2.2 interac3enetwerken .................................................................... 138
3.7.2.3 structuuranalyse van biomoleculen ............................................ 138
3.8 MODULATIE VAN GENEXPRESSIE ............................................................. 140
3.8.1 ONDERDRUKKING VAN GENEXPRESSIE VIA ANTISENSE TECHNOLOGIE ................ 140
3.8.1.1 An3sense oligonucelo3de ........................................................... 140
3.8.1.2 RNA interferen3e (RNAi) ............................................................. 141
3.8.2 GENDISRUPTIE, MUTAGENESE EN EDITING .................................................. 146
3.8.2.1 transposons ................................................................................. 146
3.8.2.4 CRISPR genoommodifica3e ......................................................... 151
3.8.3 ECTOPISCHE EXPRESSIE EN OVEREXPRESSIE ................................................ 161
3.8.3.1 directe mRNA injec3e.................................................................. 161
3.8.3.2 DNA vectoren .............................................................................. 162
3.8.4 GERICHTE PROTEÏNE DEGRADATIE ............................................................. 166
3.8.4.1 Condi3onele degrons .................................................................. 166
... 168
3
, ... 168
3.8.4.2 Bifunc3onele moleculen ............................................................. 168
3.8.4.3 TRIM away ................................................................................... 169
3.8.5 METHODEN VAN GENTRANSFER ............................................................... 169
3.8.5.1 transfec3e ................................................................................... 169
3.8.5.2 Transduc3e .................................................................................. 172
4
