De ''Moleculaire Biologie samenvatting'', is een uitgebreide samenvatting van het vak moleculaire biologie van de opleiding Life Sciences. De stof is zo duidelijk mogelijk uitgelegd met bijbehorende plaatjes, de samenvatting is uitgewerkt volgens de leerdoelen voor het tentamen, met behulp van de b...
Summary Molecular Biology of the Cell 2 (book) (WBFA007-04)
Summary Regenerative Medicine
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Hogeschool Utrecht (HU)
Biologie en Medisch Laboratoriumonderzoek
Moleculaire Biologie (TLSCMOLBI3V20)
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Moleculaire Biologie
Life Sciences, Jaar 2
Leerdoelen:
Uitleggen van de structuur van DNA en RNA in relatie tot verschillende moleculair
biologische technieken.
Toepassen van de mechanismen van replicatie op verschillende situaties in de
praktijk
Toepassen van de mechanismen van transcriptie op verschillende situaties in de
praktijk
Toepassen van de mechanismen van translatie op verschillende situaties in de
praktijk
Beschrijven van het verband tussen de opbouw van het genoom en genexpressie
Analyseren van de gevolgen van mutagenese en de toepassing hiervan in
verschillende technieken.
Analyseren en ontwerpen van een kloneer strategie.
Inhoud
Table of Contents
Inhoud....................................................................................................................................................1
Het DNA..................................................................................................................................................4
DNA structuur.....................................................................................................................................4
Het centrale dogma............................................................................................................................9
Genoomverpakking..........................................................................................................................10
Methylering en acetylering...............................................................................................................12
DNA Replicatie......................................................................................................................................13
Replicatie bubble en ORI (pro- en eukaryoten)................................................................................13
Initiatie prokaryoten.....................................................................................................................15
Initiatie Eukaryoten......................................................................................................................16
DNA polymerase...............................................................................................................................18
5’ – 3’ Exonuclease activiteit van DNA polymerases.....................................................................19
Sliding clamp.................................................................................................................................22
Helicase............................................................................................................................................22
DNA manipulation............................................................................................................................22
Restrictie enzymen...........................................................................................................................23
Ligatie...............................................................................................................................................28
Topoisomerase.................................................................................................................................29
Telomerase.......................................................................................................................................29
,Moleculair kloneren.............................................................................................................................31
PCR: polymerase chain reaction.......................................................................................................31
Primers.............................................................................................................................................32
Priming & primer verwijdering bij prokaryoten............................................................................32
Priming & primer verwijdering bij eukaryoten.............................................................................33
Primer design................................................................................................................................35
Maken van complementair DNA (cDNA)......................................................................................39
Multiple cloning site (MCS)...............................................................................................................41
Kloneringsvector...........................................................................................................................42
Restrictie enzymen.......................................................................................................................42
Transformatie...................................................................................................................................48
Heat shock transformatie.............................................................................................................48
Bacteriekweek (selectie)...................................................................................................................49
Transcriptie Prokaryoot........................................................................................................................53
Algemeen..........................................................................................................................................53
Initiatie.............................................................................................................................................54
Elongatie...........................................................................................................................................55
Terminatie........................................................................................................................................56
LAC en tryptopfaan operon..............................................................................................................58
Transcriptie Eukaryoten........................................................................................................................61
Algemeen..........................................................................................................................................61
Initiatie.............................................................................................................................................61
Eukaryote Promotors....................................................................................................................62
Algemene transcriptie factoren....................................................................................................63
Elongatie...........................................................................................................................................65
Pre-mRNA processing.......................................................................................................................65
Verschillen pro- en eukaryoot mRNA...........................................................................................66
CTD van RNApolII...........................................................................................................................66
5’- Cap...........................................................................................................................................67
Poly-A-staart.................................................................................................................................68
mRNA splicing...............................................................................................................................69
SR eiwitten....................................................................................................................................72
rRNA Processing Eukaryoten............................................................................................................72
rRNA Processing Prokaryoten...........................................................................................................73
Het gencontrolegebied.................................................................................................................73
Het mediator complex..................................................................................................................75
Eukaryotische transcriptie regelgevers verzamelen zich in complexen op...................................75
DNA..............................................................................................................................................75
, Structuur van twee humane genen die de rangschikking van exons en intronen.........................77
Translatie..............................................................................................................................................77
Eukaryoot versus prokaryoot........................................................................................................77
Translatie: Initiatie prokaryoot.........................................................................................................78
Translatie: initiatie eukaryoot...........................................................................................................79
Elongatie in pro- en eukaryoot.........................................................................................................80
Terminatie in pro- en eukaryoot.......................................................................................................81
Translatie factoren............................................................................................................................81
Wobbling......................................................................................................................................82
Isoaccepting..................................................................................................................................83
Co translationele eiwitvouwing........................................................................................................84
Eiwitten structuren.......................................................................................................................86
Prokaryoten mRNA afbraak process.................................................................................................92
mRNA afbraak Eukaryoot.................................................................................................................93
RNAi..................................................................................................................................................94
Replicatie/Transcriptie/Translatie/Elongatie/Terminatie pro- & eukaryoot samengevat....................96
Eiwit afbraak.....................................................................................................................................99
Reverse genetics.............................................................................................................................101
Knock out en knockdown............................................................................................................102
Knocdown m.b.v. siRNa..............................................................................................................103
Overexpressie.............................................................................................................................104
Bioluminescence imaging (=licht geven) met een luciferase reportergen..................................106
Genoomvariatie..............................................................................................................................107
(Post-) replicatief herstel van DNA.................................................................................................114
Pre-replicatief herstel van DNA......................................................................................................116
Mutagenese detecteren.....................................................................................................................121
Probe hybridizatie..........................................................................................................................121
Southern Blot..............................................................................................................................122
FISH.............................................................................................................................................123
Sequencing.....................................................................................................................................124
Sanger sequencing......................................................................................................................124
Next-Gen Sequencing.................................................................................................................125
Illumina.......................................................................................................................................125
Nanopore....................................................................................................................................126
Assembly van grote genomen....................................................................................................127
Mutagenese samengevat...............................................................................................................129
Site-directed mutagenesis..............................................................................................................130
Gene editing met CRISPRS / Cas9...................................................................................................132
Het DNA
DNA structuur
Het is belangrijk om te weten wat de structuur van DNA is en wat dit allemaal inhoudt. DNA is
opgebouwd uit een fosfaatgroep dat gekoppeld is aan een suiker (deoxy- of ribose). De suikergroep is
dan weer gekoppeld aan een base.
Blauw is een fosfaatgroep, zwart een suiker. De nummers in de suikergroep staan voor welke C-
atoom dit is, dit is belangrijk om de bindingen te snappen. aan C atoom 5 bindt de fosfaatgroep, wat
de backbone maakt van het DNA en op het C atoom 1 de base aan het N molecuul. De restgroep bij
nummer 2 is afhankelijk van wat het is, een ribose = OH of een deoxyribose= H. Aan het 3de
koolstofatoom bindt er een nieuwe nucleotide, hier komt een andere fosfaat groep en gaat het DNA
zo door net zoals de onderstaande afbeelding.
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