Samenvatting Moleculaire Biologie
,Inhoud
Thema 1: DNA replicatie & Moleculair kloneren............................................................................ 3
WC1 ..................................................................................................................................................... 4
Intro voorbeeld Cystic Fybrosis ....................................................................................................... 4
DNA replicatie ................................................................................................................................. 5
Moleculair kloneren (PCR) ............................................................................................................ 10
WC2 ................................................................................................................................................... 13
Initiatiefase van DNA replicatie .................................................................................................... 13
Helicases in pro- en eukaryoten ................................................................................................... 15
Moleculair kloneren (digestie en ligatie) ...................................................................................... 16
WC3 ................................................................................................................................................... 19
Elongatiefase van DNA replicatie .................................................................................................. 19
Primers verwijderen...................................................................................................................... 20
Topoisomerases ............................................................................................................................ 20
Terminatiefase van DNA replicatie ............................................................................................... 21
Moleculair kloneren (transformatie en bacteriekweek)............................................................... 22
Thema 2: Transcriptie & mRNA expressie analyse ....................................................................... 24
WC4 Prokaryote transcriptie ............................................................................................................ 25
Moleculair kloneren (bacteriekweek) ........................................................................................... 28
WC5 Eukaryote transcriptie .............................................................................................................. 29
WC6 Epigenetica & genexpressie ..................................................................................................... 34
WC7 mRNA stabiliteit en technieken................................................................................................ 42
Thema 3: Translatie & Reverse genetics ...................................................................................... 47
WC8 Translatie .................................................................................................................................. 48
WC9 Eiwitten & Reverse genetics ..................................................................................................... 53
Thema 4: Mutagenese & Identificatie van mutaties .................................................................... 61
WC11 ................................................................................................................................................. 62
Mutagenese .................................................................................................................................. 62
DNA repair..................................................................................................................................... 66
Mutaties opsporen........................................................................................................................ 70
WC12 ................................................................................................................................................. 73
Mutaties opsporen vervolg ........................................................................................................... 73
Reverse Genetics........................................................................................................................... 76
2|Page
,Thema 1: DNA replicatie & Moleculair kloneren
3|Page
, WC1
Hoofdstukken in ‘Molecular Biology of the cell’’:
H1 blz 2-7, 22-23, 25-29
H2 blz 100-101
H4 blz 175-201
H8 blz 473-474
Intro voorbeeld Cystic Fybrosis
Normaal functioneren van CFTR:
CFTR eiwit
• Cystic fibrosis transmembrane conductance regulator
• Cl- transporter in plasmamembraan van epitheliale cellen
• Actief Cl- transport reguleert passief Na+ en H20 transport over het plasma membraan:
Cl- export → Na+ export → H2O export
• Belangrijk voor hydratatie van mucuslaag in de longen en functioneren daarvan als
verdediging tegen infecties → Vloeibaar waardoor gevangen bacteriën richting de mond
kunnen worden getransporteerd
Verminderde werking CFTR:
Gemuteerd CFTR eiwit
• Verminderd Cl- export → verhoogde Na+ resorptie → dehydratatie van mucuslaag want
water gaat weg van de muscuslaag de intracellulaire ruimte in → infecties
• Mutaties geven ook fenotype in andere organen (pancreas, zweetklier)
Gemuteerd CFTR is autosomaal recessief. Er zijn enorm veel soorten mutaties die nog besproken
gaan worden.
4|Page
,Inhoud
Thema 1: DNA replicatie & Moleculair kloneren............................................................................ 3
WC1 ..................................................................................................................................................... 4
Intro voorbeeld Cystic Fybrosis ....................................................................................................... 4
DNA replicatie ................................................................................................................................. 5
Moleculair kloneren (PCR) ............................................................................................................ 10
WC2 ................................................................................................................................................... 13
Initiatiefase van DNA replicatie .................................................................................................... 13
Helicases in pro- en eukaryoten ................................................................................................... 15
Moleculair kloneren (digestie en ligatie) ...................................................................................... 16
WC3 ................................................................................................................................................... 19
Elongatiefase van DNA replicatie .................................................................................................. 19
Primers verwijderen...................................................................................................................... 20
Topoisomerases ............................................................................................................................ 20
Terminatiefase van DNA replicatie ............................................................................................... 21
Moleculair kloneren (transformatie en bacteriekweek)............................................................... 22
Thema 2: Transcriptie & mRNA expressie analyse ....................................................................... 24
WC4 Prokaryote transcriptie ............................................................................................................ 25
Moleculair kloneren (bacteriekweek) ........................................................................................... 28
WC5 Eukaryote transcriptie .............................................................................................................. 29
WC6 Epigenetica & genexpressie ..................................................................................................... 34
WC7 mRNA stabiliteit en technieken................................................................................................ 42
Thema 3: Translatie & Reverse genetics ...................................................................................... 47
WC8 Translatie .................................................................................................................................. 48
WC9 Eiwitten & Reverse genetics ..................................................................................................... 53
Thema 4: Mutagenese & Identificatie van mutaties .................................................................... 61
WC11 ................................................................................................................................................. 62
Mutagenese .................................................................................................................................. 62
DNA repair..................................................................................................................................... 66
Mutaties opsporen........................................................................................................................ 70
WC12 ................................................................................................................................................. 73
Mutaties opsporen vervolg ........................................................................................................... 73
Reverse Genetics........................................................................................................................... 76
2|Page
,Thema 1: DNA replicatie & Moleculair kloneren
3|Page
, WC1
Hoofdstukken in ‘Molecular Biology of the cell’’:
H1 blz 2-7, 22-23, 25-29
H2 blz 100-101
H4 blz 175-201
H8 blz 473-474
Intro voorbeeld Cystic Fybrosis
Normaal functioneren van CFTR:
CFTR eiwit
• Cystic fibrosis transmembrane conductance regulator
• Cl- transporter in plasmamembraan van epitheliale cellen
• Actief Cl- transport reguleert passief Na+ en H20 transport over het plasma membraan:
Cl- export → Na+ export → H2O export
• Belangrijk voor hydratatie van mucuslaag in de longen en functioneren daarvan als
verdediging tegen infecties → Vloeibaar waardoor gevangen bacteriën richting de mond
kunnen worden getransporteerd
Verminderde werking CFTR:
Gemuteerd CFTR eiwit
• Verminderd Cl- export → verhoogde Na+ resorptie → dehydratatie van mucuslaag want
water gaat weg van de muscuslaag de intracellulaire ruimte in → infecties
• Mutaties geven ook fenotype in andere organen (pancreas, zweetklier)
Gemuteerd CFTR is autosomaal recessief. Er zijn enorm veel soorten mutaties die nog besproken
gaan worden.
4|Page
