Methoden in biomedisch
onderzoek 3
Table of Contents
AFKORTINGENLIJST......................................................................................3
1. INLEIDING EN SITUERING..................................................................... 4
2. ANALYSE DNA EN GENOMICS............................................................... 5
1. DNA SEQUENTIEBEPALING.........................................................................5
1. 1E GENERATIE SEQBEPALING........................................................................................... 6
2. 2E GENERATIE SEQBEPALING (SHORT-READS NGS).............................................................6
3. 3E GENERATIE SEQBEPALING (LONG-READ)........................................................................9
4. EPIGENITICA SEQBEPALING........................................................................................... 10
5. SINGLE-CELL SEQUENCING........................................................................................... 11
2. DNA GENOTYPEREN...............................................................................11
1. SHORT TANDEM REPEAT GENOTYPERING.........................................................................11
2. TAQMAN GENOTYPERING............................................................................................. 11
3. MICROARRAYS........................................................................................................... 12
3. DNA HYBRIDISEREN...............................................................................12
1. FISH...................................................................................................................... 12
2. CGH...................................................................................................................... 12
SNP MICRO-ARRAYS TOEGEPAST OP CNV’S........................................................................12
3. ANALYSE RNA EN TRANSCRIPTOMICS................................................. 12
1) RNA MICROARRAYS...............................................................................13
2) RNA SEQUENCING (BULK RNA SEQ)..........................................................14
1) SHORT-READ CDNA SEQUENCING: 2E GS.....................................................................15
2) LONG-READ CDNA (3E GS) EN DIRECT RNA SEQUENCING...............................................16
3) SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ)...............................................................16
4. SPATIAL TRANSCRIPTOMICS.......................................................................................... 17
4. ANALYSE EIWITTEN EN PROTEOMICS.................................................. 18
1) EIWITSEQUENTIEBEPALING (ID).................................................................18
1. EDMANDEGRADATIE................................................................................................... 18
2. EIWITSEQUENTIEBEPALING VIA MS................................................................................ 18
,2) PROTEOOMANALYSE...............................................................................19
1. BULK ANALYSE: PROTEOMICS OP EIWITEXTRACTEN............................................................19
2. SINGLE CELL PROTEOMICS........................................................................................... 21
3. SPATIAL PROTEOMICS................................................................................................. 21
3) 3D-STRUCTUURBEPALING........................................................................22
1. X-STRAALDIFFRACTIE EN KRISTALLOGRAFIE......................................................................22
2. NMR SPECTROSCOPIE................................................................................................ 23
3. CRYO-EM................................................................................................................ 23
4. EIWIT-EIWIT EN EIWIT-LIGAND INTERACTIES.....................................................................24
5. ANALYSE METABOLIETEN EN LIPIDEN.................................................. 26
1) SPECIFIEKE TESTEN INDIVIDUELE METABOLIETEN.............................................26
2) METABOLOMICS....................................................................................26
3) FLUXOMICS: METABOLE FLUXANALYSE.........................................................27
4) LIPIDOMICS.........................................................................................28
6. ANALYSE GENTRANSCRIPTIE.............................................................. 31
1. NASCENT RNA ANALYSES........................................................................31
RUN-ON ESSAYS (RO-SEQ); BASIC RUN-ON......................................................................31
ANDERE NASCENT RNA ANALYSE METHODEN......................................................................32
IMAGING-BASED NASCENT RNA ANALYSE METHODEN............................................................32
2. PROMOTER REPORTER STUDIES..................................................................32
3. ANALYSE DNA-EIWIT INTERACTIES.............................................................33
7. DATA-ANALYSE EN DATABANKEN........................................................ 35
8. MODULATIE GENEXPRESSIE............................................................... 38
1. ONDERDRUKKING VAN GENEXPRESSIE VIA ANTISENSE TECH................................38
ASO’S........................................................................................................................ 38
RNAI.......................................................................................................................... 38
2. GENDISRUPTIE, MUTAGENESE, EDITING........................................................39
TRANSPOSONS.............................................................................................................. 39
SITE-SPECIFIEKE DNA-RECOMBINATIE................................................................................40
MUTAGENESE............................................................................................................... 41
CRISPR GENOOMMODIFICATIE......................................................................................... 41
3. ECTOPISCHE EXPRESSIE EN OVEREXPRESSIE...................................................44
1) DIRECTE MRNA INJECTIE............................................................................................ 44
2) DNA VECTOREN....................................................................................................... 44
4. GERICHTE PROTEÏNE-DEGRADATIE...............................................................44
,1) CONDITIONELE DEGRONS............................................................................................ 45
2) BIFUNCTIONELE MOLECULEN........................................................................................ 45
3) TRIM AWAY.............................................................................................................. 45
5. METHODEN VAN GENTRANSFER..................................................................45
1) TRANSFECTIE............................................................................................................ 45
2) TRANSDUCTIE........................................................................................................... 46
9. INLEIDING SYSTEEMBIOLOGIE EN SYNTHETISCHE BIOLOGIE.................47
1) SYSTEEMBIOLOGIE.................................................................................47
2) SYNTHETISCHE BIO................................................................................48
10. METHODE-INTEGRATIE, OPLOSSEN CONCRETE BIOMEDISCHE
VRAAGSTELLINGEN............................................................................... 48
Afkortingenlijst
AAV: Adeno-Associated Virus
ASO: AntiSense Oligonucleotide
ATAC-seq: Assay for Transposase-Accessible Chromatin (met high-throughput
seq)
bDNA Branched DNA
BiFC Bimoleculaire Fluorescentie Complementatie
CITE: Cellular Indexing of Transcriptomes and Epitopes
CGH: Comparative Genomic Hybridization
ChIP Chromatine ImmunoPrecipitatie
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
ECAR: ExtraCellular Acidification Rate
EMSA: Electrophoretic Mobility Shift Assay
FAIRE(-seq): Formaldehyde-Assisted Isolation of Regulatory Elements
GEM Gel beads in emulsion
GRO-Seq: Global Run-On-sequencing
GSEA: Gene set enrichment analysis
GWAS: genoomwijde associatiestudies
HDR: Homology Directed Repair
IRES Internal Ribosome Entry Site
MAD: Multiwavelength Anomalous Dispersion
MALBAC: Multiple Annealing and Looping Based Amplification Cycles
MALDI: Matrix-Assisted Laser Desorption/Ionization
MDA: Multiple Displacement Amplification
MERFISH: Multiplex Error Robust FISH
MIR: Multiple Isomorphous Replacement
MOE Model Organism Ecogenomix
, MR: Molecular Replacement
MRM: Multiple Reaction Monitoring
NGS: Next generation sequencing
NHEJ: Non-Homologous End-Joining
NMR: Nuclear Magnetic Resonance
NIPT: Niet-invasieve prenatale test
OCR: Oxygen consumption rate
PAM: Protospacer Adjacent Motif
PB: Piggy Bac
PCA: Principal Component Analysis (RNA)
PMF: Peptide Mass Fingerprint
PRM: Parallel Reaction Monitoring
PRO-Seq: Precision nuclear Run-On sequencing
PROTAC: Proteolysis-Targeting Chimeric molecule
RCA: Rolling Circle Amplification
(F/B)RET: Resonance Energy Transfer
RACE Rapid Amplification Cdna Ends
RIME: Rapid Immunoprecipitation MS of Endogenous proteins
SB: Sleeping Beauty
SBS: Sequencing By Synthesis
sciMET: Single cell Combinatorial Indexing METhyl-seq
SCI-seq: Single-cell combinatorial indexed sequencing
shRNA: short hairpin RNA
SIRM: Stable Isotope-Resolved Metabolomics
siRNA: small interfering RNA
SLIC: Seq Ligation Independent Cloning
smFISH: single molecule Fluo In Situ Hybridisation
SMRT: Single-Molecule Real-Time
SNIPER: Specific Non-genetic Inhibitor of apoptosis protein (IAP)-dependent
P-erasers
SOMAmer: Slow Off-rate Modified Aptamer
SPR: Surface Plasmon Resonance
SRM: Selected Reaction Monitoring
TTO Terminal tagging oligo
UMI: Unique Molecular Identifier
1. Inleiding en situering
24 lessen 1,5u – aandacht op onderzoeksstrategie leren opstellen, overzicht
bewaren; principes begrijpen, strategische keuze leren maken, inzicht in
belang en impact…
Examen: 20 MC-vragen (10), 3 open vragen (6), vraagstuk (4)
onderzoek 3
Table of Contents
AFKORTINGENLIJST......................................................................................3
1. INLEIDING EN SITUERING..................................................................... 4
2. ANALYSE DNA EN GENOMICS............................................................... 5
1. DNA SEQUENTIEBEPALING.........................................................................5
1. 1E GENERATIE SEQBEPALING........................................................................................... 6
2. 2E GENERATIE SEQBEPALING (SHORT-READS NGS).............................................................6
3. 3E GENERATIE SEQBEPALING (LONG-READ)........................................................................9
4. EPIGENITICA SEQBEPALING........................................................................................... 10
5. SINGLE-CELL SEQUENCING........................................................................................... 11
2. DNA GENOTYPEREN...............................................................................11
1. SHORT TANDEM REPEAT GENOTYPERING.........................................................................11
2. TAQMAN GENOTYPERING............................................................................................. 11
3. MICROARRAYS........................................................................................................... 12
3. DNA HYBRIDISEREN...............................................................................12
1. FISH...................................................................................................................... 12
2. CGH...................................................................................................................... 12
SNP MICRO-ARRAYS TOEGEPAST OP CNV’S........................................................................12
3. ANALYSE RNA EN TRANSCRIPTOMICS................................................. 12
1) RNA MICROARRAYS...............................................................................13
2) RNA SEQUENCING (BULK RNA SEQ)..........................................................14
1) SHORT-READ CDNA SEQUENCING: 2E GS.....................................................................15
2) LONG-READ CDNA (3E GS) EN DIRECT RNA SEQUENCING...............................................16
3) SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ)...............................................................16
4. SPATIAL TRANSCRIPTOMICS.......................................................................................... 17
4. ANALYSE EIWITTEN EN PROTEOMICS.................................................. 18
1) EIWITSEQUENTIEBEPALING (ID).................................................................18
1. EDMANDEGRADATIE................................................................................................... 18
2. EIWITSEQUENTIEBEPALING VIA MS................................................................................ 18
,2) PROTEOOMANALYSE...............................................................................19
1. BULK ANALYSE: PROTEOMICS OP EIWITEXTRACTEN............................................................19
2. SINGLE CELL PROTEOMICS........................................................................................... 21
3. SPATIAL PROTEOMICS................................................................................................. 21
3) 3D-STRUCTUURBEPALING........................................................................22
1. X-STRAALDIFFRACTIE EN KRISTALLOGRAFIE......................................................................22
2. NMR SPECTROSCOPIE................................................................................................ 23
3. CRYO-EM................................................................................................................ 23
4. EIWIT-EIWIT EN EIWIT-LIGAND INTERACTIES.....................................................................24
5. ANALYSE METABOLIETEN EN LIPIDEN.................................................. 26
1) SPECIFIEKE TESTEN INDIVIDUELE METABOLIETEN.............................................26
2) METABOLOMICS....................................................................................26
3) FLUXOMICS: METABOLE FLUXANALYSE.........................................................27
4) LIPIDOMICS.........................................................................................28
6. ANALYSE GENTRANSCRIPTIE.............................................................. 31
1. NASCENT RNA ANALYSES........................................................................31
RUN-ON ESSAYS (RO-SEQ); BASIC RUN-ON......................................................................31
ANDERE NASCENT RNA ANALYSE METHODEN......................................................................32
IMAGING-BASED NASCENT RNA ANALYSE METHODEN............................................................32
2. PROMOTER REPORTER STUDIES..................................................................32
3. ANALYSE DNA-EIWIT INTERACTIES.............................................................33
7. DATA-ANALYSE EN DATABANKEN........................................................ 35
8. MODULATIE GENEXPRESSIE............................................................... 38
1. ONDERDRUKKING VAN GENEXPRESSIE VIA ANTISENSE TECH................................38
ASO’S........................................................................................................................ 38
RNAI.......................................................................................................................... 38
2. GENDISRUPTIE, MUTAGENESE, EDITING........................................................39
TRANSPOSONS.............................................................................................................. 39
SITE-SPECIFIEKE DNA-RECOMBINATIE................................................................................40
MUTAGENESE............................................................................................................... 41
CRISPR GENOOMMODIFICATIE......................................................................................... 41
3. ECTOPISCHE EXPRESSIE EN OVEREXPRESSIE...................................................44
1) DIRECTE MRNA INJECTIE............................................................................................ 44
2) DNA VECTOREN....................................................................................................... 44
4. GERICHTE PROTEÏNE-DEGRADATIE...............................................................44
,1) CONDITIONELE DEGRONS............................................................................................ 45
2) BIFUNCTIONELE MOLECULEN........................................................................................ 45
3) TRIM AWAY.............................................................................................................. 45
5. METHODEN VAN GENTRANSFER..................................................................45
1) TRANSFECTIE............................................................................................................ 45
2) TRANSDUCTIE........................................................................................................... 46
9. INLEIDING SYSTEEMBIOLOGIE EN SYNTHETISCHE BIOLOGIE.................47
1) SYSTEEMBIOLOGIE.................................................................................47
2) SYNTHETISCHE BIO................................................................................48
10. METHODE-INTEGRATIE, OPLOSSEN CONCRETE BIOMEDISCHE
VRAAGSTELLINGEN............................................................................... 48
Afkortingenlijst
AAV: Adeno-Associated Virus
ASO: AntiSense Oligonucleotide
ATAC-seq: Assay for Transposase-Accessible Chromatin (met high-throughput
seq)
bDNA Branched DNA
BiFC Bimoleculaire Fluorescentie Complementatie
CITE: Cellular Indexing of Transcriptomes and Epitopes
CGH: Comparative Genomic Hybridization
ChIP Chromatine ImmunoPrecipitatie
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
ECAR: ExtraCellular Acidification Rate
EMSA: Electrophoretic Mobility Shift Assay
FAIRE(-seq): Formaldehyde-Assisted Isolation of Regulatory Elements
GEM Gel beads in emulsion
GRO-Seq: Global Run-On-sequencing
GSEA: Gene set enrichment analysis
GWAS: genoomwijde associatiestudies
HDR: Homology Directed Repair
IRES Internal Ribosome Entry Site
MAD: Multiwavelength Anomalous Dispersion
MALBAC: Multiple Annealing and Looping Based Amplification Cycles
MALDI: Matrix-Assisted Laser Desorption/Ionization
MDA: Multiple Displacement Amplification
MERFISH: Multiplex Error Robust FISH
MIR: Multiple Isomorphous Replacement
MOE Model Organism Ecogenomix
, MR: Molecular Replacement
MRM: Multiple Reaction Monitoring
NGS: Next generation sequencing
NHEJ: Non-Homologous End-Joining
NMR: Nuclear Magnetic Resonance
NIPT: Niet-invasieve prenatale test
OCR: Oxygen consumption rate
PAM: Protospacer Adjacent Motif
PB: Piggy Bac
PCA: Principal Component Analysis (RNA)
PMF: Peptide Mass Fingerprint
PRM: Parallel Reaction Monitoring
PRO-Seq: Precision nuclear Run-On sequencing
PROTAC: Proteolysis-Targeting Chimeric molecule
RCA: Rolling Circle Amplification
(F/B)RET: Resonance Energy Transfer
RACE Rapid Amplification Cdna Ends
RIME: Rapid Immunoprecipitation MS of Endogenous proteins
SB: Sleeping Beauty
SBS: Sequencing By Synthesis
sciMET: Single cell Combinatorial Indexing METhyl-seq
SCI-seq: Single-cell combinatorial indexed sequencing
shRNA: short hairpin RNA
SIRM: Stable Isotope-Resolved Metabolomics
siRNA: small interfering RNA
SLIC: Seq Ligation Independent Cloning
smFISH: single molecule Fluo In Situ Hybridisation
SMRT: Single-Molecule Real-Time
SNIPER: Specific Non-genetic Inhibitor of apoptosis protein (IAP)-dependent
P-erasers
SOMAmer: Slow Off-rate Modified Aptamer
SPR: Surface Plasmon Resonance
SRM: Selected Reaction Monitoring
TTO Terminal tagging oligo
UMI: Unique Molecular Identifier
1. Inleiding en situering
24 lessen 1,5u – aandacht op onderzoeksstrategie leren opstellen, overzicht
bewaren; principes begrijpen, strategische keuze leren maken, inzicht in
belang en impact…
Examen: 20 MC-vragen (10), 3 open vragen (6), vraagstuk (4)
