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Methoden 3 - samenvatting

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Course
Methoden in het biomedisch onderzoek 3 (E0F95A)

Institution
Katholieke Universiteit Leuven (KU Leuven)

Samenvatting van notities en slides voor het vak Methoden in het Biomedisch onderzoek 3 gegeven door Daelemans Dirk | De Keersmaecker Kim | Goris An | Swinnen Johan. Door dit document te studeren behaalde in een 15/20 in eerste zit.

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Uploaded on January 18, 2025
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Number of pages 119
Written in 2022/2023
Type Summary

dna genomics
transcriptomics
methoden 3
proteomics
metabolomics
gentranscriptie
databanken data analyse
modulatie genexpressie
systeembiologie

Institution
Katholieke Universiteit Leuven (KU Leuven)
Education
Biomedische Wetenschappen
Course
Methoden in het biomedisch onderzoek 3 (E0F95A)

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Methoden 3
INHOUD

1. Inleiding ............................................................................................................................................................. 4
2. Analyse van DNA en genomics .......................................................................................................................... 4
2.1. DNA sequentiebepaling ................................................................................................................................ 4
2.1.1. 1ste generatie sequentiebepaling .......................................................................................................... 5
2.1.2. 2de generatie sequentiebepaling ........................................................................................................... 7
2.1.3. 3de generatie sequentiebepaling: long-read next-generation sequencing ......................................... 15
2.1.4. Epigenetica sequentiebepaling (methylatie sequentiebepaling) ........................................................ 18
2.1.5. Single-cell sequentiebepaling.............................................................................................................. 20
2.2. DNA genotyperen ....................................................................................................................................... 21
2.2.1. Short-tandem repeat (STR) genotypering ........................................................................................... 22
2.2.2. Taqman genotypering ......................................................................................................................... 22
2.2.3. Microarrays (+ genoomwijde associatiestudies) ................................................................................. 23
2.3. DNA hybridiseren ....................................................................................................................................... 25
2.3.1. FISH ..................................................................................................................................................... 25
2.3.2. CGH ..................................................................................................................................................... 26
2.3.3. SNP micro-arrays toegepast op CNV’s ................................................................................................ 26
3. Analyse van RNA en transcriptomics ............................................................................................................... 28
3.1. RNA micro-ARRAYS..................................................................................................................................... 28
3.1.1. Voorbeelden ........................................................................................................................................ 31
3.2. RNA sequencing (bulk) ............................................................................................................................... 31
3.2.1. short-read RNA (cDNA) sequencing .................................................................................................... 32
3.2.2. long-read cDNA en direct RNA sequencing ......................................................................................... 34
3.3. Single-cell RNA sequencing ........................................................................................................................ 34
3.3.1. Droplet sequencing ............................................................................................................................. 34
3.3.2. CITE-seq ............................................................................................................................................... 35
3.4. Spatial transcriptomics ............................................................................................................................... 36
4. Analyse van eiwitten en proteomics ............................................................................................................... 40
4.1. Sequentiebepaling ..................................................................................................................................... 40
4.1.1. Edman degradatie ............................................................................................................................... 40
4.1.2. Eiwit sequentie bepaling via massa spectrometrie ............................................................................. 40
4.2. Proteoom analyse ...................................................................................................................................... 41
4.2.1. Bulk analyse......................................................................................................................................... 41
4.2.2. Single cell proteomics ......................................................................................................................... 45
4.2.3. Spatial proteomics............................................................................................................................... 46
4.3. 3D structuur bepaling................................................................................................................................. 48
4.3.1. X-straaldiffreactie en kristallografie............................................................................................ 48
4.3.2. NMR spectroscopie ............................................................................................................................. 52

1

, 4.3.3. Cryo-elektronenmicroscopie – Cryo-EM ............................................................................................. 52
4.3.4. Overzicht van methodes voor eiwitstructuur ..................................................................................... 53
4.4. Eiwit-eiwit interacties................................................................................................................................. 53
4.4.1. Yeast-2-hybrid en mammalian 2-hybrid ............................................................................................ 53
4.4.2. Affinity pull-down .............................................................................................................................. 55
4.4.3. Co-immunoprecipitatie (co-IP) .......................................................................................................... 55
4.4.4. Fluorescence Resonance Energy Transfer (FRET) & Bioluminiscence Resonance Energy Transfer
(BRET) ............................................................................................................................................................ 56
4.4.5. Bindingsarrays ................................................................................................................................... 57
4.4.6. Surface plasmon resonance .............................................................................................................. 57
4.4.7. Phage display ...................................................................................................................................... 58
4.4.8. Proximity Ligation Assay (PLA) ........................................................................................................... 58
4.4.9. RIME ................................................................................................................................................... 59
4.4.10. Ligand-receptor bindingsstudies ....................................................................................................... 60
5. Analyse van metabolieten en lipiden .............................................................................................................. 62
5.1. Specifieke testen van individuele metabolieten ........................................................................................ 62
5.2. Metabolomics ............................................................................................................................................ 62
5.2.1. Via NMR-spectroscopie ....................................................................................................................... 63
5.2.2. Via MassaSpectrometrie ..................................................................................................................... 63
5.3. Metabole flux analyse ................................................................................................................................ 64
5.3.1. Stable isotope-resolved metabolomics (SIRM) /Tracer metabolomics (via NMR of MS) ................... 64
5.3.2. Metabolic flux analyzer (Seahorse) ..................................................................................................... 65
5.4. Analyse van lipiden..................................................................................................................................... 66
5.4.1. Extractie van LIPIDEN .......................................................................................................................... 66
5.4.2. Thin layer chromatografie - TLC / high performance TLC / 2D-TLC ..................................................... 67
5.4.3. High performance liquid chromatografie - HPLC ................................................................................ 67
5.4.4. Gaschromatografie - GC ...................................................................................................................... 68
5.4.5. MS-based lipidomics ........................................................................................................................... 68
5.4.6. Spatial lipidomics ................................................................................................................................ 71
6. Analyse van gentranscriptie ............................................................................................................................ 74
6.1. Nascent RNA analyses ................................................................................................................................ 74
6.1.1. Run on ASSAYS .................................................................................................................................... 74
6.1.2. Andere nascent RNA analyse methoden ............................................................................................. 75
6.1.3. Imaging-gebaseerde nascent RNA analyse methoden ........................................................................ 76
6.2. Promoter-reporter studies ......................................................................................................................... 77
6.3. Analyse van protein-DNA interacties ......................................................................................................... 77
6.3.1. DNase footprinting .............................................................................................................................. 77
6.3.2. EMSA ................................................................................................................................................... 78
6.3.3. Open chromatine ASSAYS ................................................................................................................... 79
6.3.4. Chromatine immunoprecipitatie ASSAYS ............................................................................................ 80
7. Databanken en data-analyse ........................................................................................................................... 82

2

, 7.1. Databanken ................................................................................................................................................ 82
7.1.1. Literatuur ............................................................................................................................................ 82
7.1.2. Data repositoria .................................................................................................................................. 82
7.1.3. Data platformen .................................................................................................................................. 83
7.2. Data analyse ............................................................................................................................................... 84
7.2.1. Annotatie van genlijsten ..................................................................................................................... 84
7.2.2. Interactienetwerken............................................................................................................................ 85
7.2.3. Structuuranalyse van biomoleculen .................................................................................................... 85
8. Modulatie van genexpressie ........................................................................................................................... 86
8.1. Onderdrukking van genexpressie via antisense technologie ..................................................................... 86
8.1.1. Antisense oligonucleotiden (ASO) .............................................................................................. 86
8.1.2. RNA interference (RNAi) ............................................................................................................. 87
8.2. Gendisruptie, mutagenese en editing ........................................................................................................ 89
8.2.1. Transposons ................................................................................................................................ 89
8.2.2. Site specific DNA recombinatie ................................................................................................... 92
8.2.3. Mutagenese ................................................................................................................................ 94
8.2.4. CRISPR ......................................................................................................................................... 94
8.3. Ectopische expressie en overexpressie .................................................................................................... 100
8.3.1. Directe injectie van mRNA ........................................................................................................ 100
8.3.2. DNA vectoren .................................................................................................................................... 100
8.4. Gerichte proteïne degradatie ................................................................................................................... 103
8.4.1. Conditionele degrons ................................................................................................................ 103
8.4.2. Bifunctionele moleculen: PROTACs .......................................................................................... 104
8.4.3. TRIM away ........................................................................................................................................ 105
8.5. Methoden van gentransfer ...................................................................................................................... 106
8.5.1. Transfectie ................................................................................................................................ 106
8.5.2. Transductie ............................................................................................................................... 108
9. Inleiding tot systeembiologie en synthetische biologie ................................................................................ 112
9.1. Systeembiologie ....................................................................................................................................... 112
9.2. Synthetische biologie ............................................................................................................................... 112
10. Integreren van methoden en oplossen van concrete biomedische vraagstellingen .................................... 113
10.1. Voorbeeld 1 ............................................................................................................................................ 113
10.2. Voorbeeld 2 ............................................................................................................................................ 115
10.3. Voorbeeld 3 ............................................................................................................................................ 116
10.4 Voorbeeld 4 ............................................................................................................................................. 117
10.5. Voorbeeld 5 ............................................................................................................................................ 118

3

,1. INLEIDING

Belang van methoden
Grote verscheidenheid vraagstellingen waarbij het antwoord afhankelijk is van de methoden die we beschikbaar
hebben/gebruiken
→ Methoden in Biomedisch onderzoek zijn essentieel voor medische vooruitgang

2. ANALYSE VAN DNA EN GENOMICS

2.1. DNA SEQUENTIEBEPALING

Eerste generatie - 1977
- Sanger, (Maxam & Gilbert)
- Goedkoper dan NGS ter controle van constructen in labo context
- Humaan genoom project, 1 euro per base, 10 jaar durend project, 1990-2003

Tweede generatie - 2006
- ‘Next Generation Sequencing’ (NGS)
- Massief parallel sequencen van clonaal in vitro geamplificeerde DNA moleculen
- Goedkoper en sneller
- Minder mensen en minder machines
- Illumina, Ion Torrent

Derde generatie - 2010
- ‘Next-next Generation Sequencing’
- Sequencing van individuele DNA moleculen (geen amplificatie nodig)
- Nog sneller, nog minder mensen, nog goedkoper
- Zeer groot en belangrijk voor de nieuwe diagnostiek
- Nanopore, Pacific Biosciences

Brede toepassingen
De novo
Genoomsequentie is nog niet gekend, onbekende genomen
Nieuwe bacterie voor de allereerste keer sequencen
Overlappende stukken nodig om zo tot totaal genoom te komen om puzzel te kunnen leggen
Moeilijker aangezien er veel repetitieve sequenties aanwezig zijn (uitdaging)

Resequencing
Referentiegenoom beschikbaar ter vergelijking met wat we zelf sequencen
Individuen t.o.v. referentie genoom
Verschillen tussen mensen wel aanwezig SNP (3 miljoen verschil op 3 miljard)
Makkelijker om puzzel te leggen, sequentie mappen op referentiegenoom
Toepassingen Klinisch: NIPT, BRCA → diagnose
Farmacogenetica: dubbel, door individuele reactie en SNP’s kan iedereen anders reageren op
medicijn en zo soms sterfgevallen

Sequentie als telling
Typische bij transcriptomics
Aantallen DNA (of RNA) moleculen
RNA naar cDNA, deze sequencen en kijken hoe vaak transcript voorkomt en bepaalde sequentie kunnen gaan
tellen
Vb RNA Seq en ChIP Seq

4

, Figuur 1: De novo versus resequencing

Whole genome, exome en targeted sequencing
WGS Whole-genome sequencing
WES Whole-exome sequencing
= coderende sequentie
= 1.5% van genoom
Targeted sequencing Specifieke gewenste regio’s
Transcriptoom RNA → cDNA

Als er fout is bij een bepaald eiwit dan kan men beter enkel het gen gaan sequencen in plaats van het volledige
genoom

2.1.1. 1 S T E GENERATIE SEQUENTIEBEPALING

Twee methoden Maxam en Gilbert (1977)
Sanger (1980)

Maxam en Gilbert
= chemische klieving methode
Niet meer gebruikt, historisch

Principe:
- Differentiële chemische klieving van nucleïnezuren in 4 verschillende reacties, gevolgd door scheiding
volgens grootte op gel en visualisatie via autoradiografie
- Radio-actief merken: dsDNA gaan merken met radioactief materiaal, P-groep op het einde is radioactief
gemaakt, een van de 2 labels weggooien door restrictie enzym→ zo dsDNA met 1 streng gemerkt,
daardoor maar 1 streng gevisualiseerd
- Chemisch afbreken door te scheiden in 4 verschillende buisjes en met reagens chemische stof die breuk
veroorzaakt na G, A+G, C+T, C, voldoende info om sequentie te kunnen bepalen, op gel, korte
fragmenten zijn het snelst zichtbaar, van beneden naar boven sequentie aflezen

5

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