Samenvatting Moleculaire genetica
Inhoud
Les 1: Erfelijk materiaal ........................................................................................................................... 9
DNA...................................................................................................................................................... 9
Chromosomen ................................................................................................................................. 9
Karyogram vd mens ......................................................................................................................... 9
Bouwstenen..................................................................................................................................... 9
Structuur ........................................................................................................................................ 10
Humaan DNA ..................................................................................................................................... 10
Uniek DNA (50%) ........................................................................................................................... 10
Repetitief DNA (50%) => satelliet .................................................................................................. 10
Verandering op de genen (Rep. DNA) ........................................................................................... 10
Samenstelling ................................................................................................................................ 11
Satelliet DNA.................................................................................................................................. 11
Waar zit DNA ..................................................................................................................................... 12
Chromosomaal DNA ...................................................................................................................... 12
Extrachromosomaal DNA: circulair ............................................................................................... 12
Mitochondriaal DNA ...................................................................................................................... 12
Les 2: Van gen naar eiwit: transcriptie .................................................................................................. 13
Inleiding ............................................................................................................................................. 13
Soorten EW........................................................................................................................................ 13
Structurele EW .............................................................................................................................. 13
Transport-EW ................................................................................................................................ 14
Bewegings-EW ............................................................................................................................... 14
Reserve-EW ................................................................................................................................... 14
Signaal- en receptor-EW ................................................................................................................ 14
Gen-regulatorische EW ................................................................................................................. 14
Beschermende EW ........................................................................................................................ 14
Het dogma van Crick ......................................................................................................................... 14
Structuur van RNA ............................................................................................................................. 15
Soorten RNA .................................................................................................................................. 15
Verschil DNA en RNA ..................................................................................................................... 15
Eukaryote genen............................................................................................................................ 15
Polymerase II genen! ..................................................................................................................... 16
1
, Structuur van een gen ....................................................................................................................... 16
Transcriptie........................................................................................................................................ 17
Transcriptiefactoren ...................................................................................................................... 17
Structuur van mRNA .......................................................................................................................... 17
PTM 1 = post transcriptionele modificatie 1 ..................................................................................... 18
Cap structuur ................................................................................................................................. 18
Spliceosoom .................................................................................................................................. 18
Alternatieve splitsing ..................................................................................................................... 18
Les 3: De weg van gen naar EW 2 .......................................................................................................... 19
Genetische code ................................................................................................................................ 19
Translatie ........................................................................................................................................... 20
tRNA............................................................................................................................................... 20
Ribosomen ..................................................................................................................................... 20
Fasen van de translatie.................................................................................................................. 21
Groeiende polypeptideketen ........................................................................................................ 22
Transcriptie en translatie in prokaryoten en eukaryoten ............................................................. 22
PTM 2 = post translationele modificatie 2 ........................................................................................ 22
Gesecreteerde EW......................................................................................................................... 22
Glycosylatie ................................................................................................................................... 23
Fosforylatie .................................................................................................................................... 23
Acetylering..................................................................................................................................... 23
Hydroxylatie van proline ............................................................................................................... 24
Partiële proteolyse ........................................................................................................................ 24
Soorten proteasen ......................................................................................................................... 24
PTM 1 vs. PTM 2 ................................................................................................................................ 24
LES 4: Regulatie van genexpressie......................................................................................................... 25
Inleiding ............................................................................................................................................. 25
Regulatie van eiwitsynthese .............................................................................................................. 25
Voorbeelden EUK-genregulatie: .................................................................................................... 26
Epigenetica ........................................................................................................................................ 27
Mechanismen ................................................................................................................................ 28
DNA methylatie: ................................................................................................................................ 28
Overerving: .................................................................................................................................... 28
Genomische inprinting: ................................................................................................................. 28
Histonmodificatie .............................................................................................................................. 29
RNA interferentie .............................................................................................................................. 29
2
, Pathway ......................................................................................................................................... 30
Dicer: ............................................................................................................................................. 30
Sequentie specifieke mRNA degradatie: ....................................................................................... 30
miRNA: ........................................................................................................................................... 30
LES 5: Celdeling en overerving van het geslacht ................................................................................... 31
Inleiding ............................................................................................................................................. 31
De celcyclus ....................................................................................................................................... 31
G1: werkingsfase ........................................................................................................................... 31
S: synthese ..................................................................................................................................... 31
G2: laatste controle voor deling .................................................................................................... 32
M: delingsfase ............................................................................................................................... 32
Replicatie ........................................................................................................................................... 32
Replicatievork ................................................................................................................................ 32
Proof reading ................................................................................................................................. 33
Leading en lagging streng synthese............................................................................................... 33
Verlies van DNA ............................................................................................................................. 34
Werking telomerase ...................................................................................................................... 34
Soort deling ....................................................................................................................................... 34
Mitose............................................................................................................................................ 34
Meiose ........................................................................................................................................... 35
Herverdeling van het erfelijk materiaal............................................................................................. 35
Crossing over ................................................................................................................................. 35
Segregatie ...................................................................................................................................... 36
De voortplantingscellen/ gameten.................................................................................................... 36
De man: ......................................................................................................................................... 36
De vrouw: ...................................................................................................................................... 37
Geslachtsbepaling bij bevruchting ................................................................................................ 37
Sexratio .......................................................................................................................................... 37
Y-chromosoom .............................................................................................................................. 38
X-chromosoom .............................................................................................................................. 38
LES 6: Mutaties ...................................................................................................................................... 39
Puntmutaties ..................................................................................................................................... 39
Sikkelcelanemie: ............................................................................................................................ 39
Voorkomen mutaties..................................................................................................................... 40
Micro-deleties en –inserties .............................................................................................................. 40
Trinucleotide repeat amplificaties (dynamische mutaties)............................................................... 40
3
, Mutaties die de transcriptie of mRNA processing verstoren ............................................................ 41
Lengtemutaties.................................................................................................................................. 42
Soorten .......................................................................................................................................... 42
Ongelijke recombinatie ................................................................................................................. 42
Translocaties.................................................................................................................................. 42
Transpositie ................................................................................................................................... 42
Oorzaken en gevolgen mutaties........................................................................................................ 43
Oorzaken spontane mutaties ........................................................................................................ 43
Externe invloeden door mutagenen.............................................................................................. 43
Gevolgen mutaties ........................................................................................................................ 43
LES 7: Monogene overerving ................................................................................................................. 44
Inleiding ............................................................................................................................................. 44
Kruiswetten van Mendel ............................................................................................................... 44
Basisbegrippen .................................................................................................................................. 45
Genotype en fenotype: ................................................................................................................. 45
Variabiliteit .................................................................................................................................... 45
Genotype naar fenotype ............................................................................................................... 45
Ontstaan ziekten ............................................................................................................................... 45
Recessieve ziekten ......................................................................................................................... 45
Dominante ziekten ........................................................................................................................ 45
Stamboom ......................................................................................................................................... 46
Autosomale overerving ..................................................................................................................... 46
Recessieve aandoeningen ............................................................................................................. 46
Dominante aandoeningen ............................................................................................................. 46
Partiële dominantie ....................................................................................................................... 47
Codominantie bij ABO bloedgroep................................................................................................ 47
LES 8: Geslachtsgebonden overerving .................................................................................................. 47
Geslachtsgebonden kenmerken ........................................................................................................ 47
X-gebonden recessieve aandoeningen ............................................................................................. 47
Stamboom ..................................................................................................................................... 47
Hemofilie/ bloederziekte............................................................................................................... 48
Duchenne(DMD) ............................................................................................................................ 48
Becker(BMD) ................................................................................................................................. 48
Fragiele X-syndroom...................................................................................................................... 48
Daltonisme .................................................................................................................................... 48
Syndroom van Lesch-Nyhan .......................................................................................................... 48
4
, Variaties in G6PDH ........................................................................................................................ 48
X-gebonden dominante aandoeningen............................................................................................. 48
Stamboom ..................................................................................................................................... 49
Syndroom van Rett (RTT)............................................................................................................... 49
Het Y-chromosoom............................................................................................................................ 49
Overerving ..................................................................................................................................... 49
Stamboom ..................................................................................................................................... 49
Mitochondriale overerving ................................................................................................................ 49
Kenmerken .................................................................................................................................... 49
Stamboom ..................................................................................................................................... 49
LES 9 : Multifactoriële overerving ......................................................................................................... 50
Inleiding ............................................................................................................................................. 50
Beïnvloeding door meerdere genen.................................................................................................. 50
Additieve polygenie ....................................................................................................................... 50
Drempel polygenie ........................................................................................................................ 50
Epistase en hypostase ................................................................................................................... 50
Beïnvloeding vanuit de omgeving ..................................................................................................... 51
Complicerende processen ................................................................................................................. 51
Locus heterogeniteit...................................................................................................................... 51
Pleiotropie ..................................................................................................................................... 51
Penetrantie en expressiviteit ........................................................................................................ 51
Mozaïken ....................................................................................................................................... 51
Genetische anticipatie ................................................................................................................... 52
Epi genetische invloeden( zie Epigenetica) ................................................................................... 52
LES 10: chromosomale afwijkingen ....................................................................................................... 52
Inleiding ............................................................................................................................................. 52
Polyploïdie ......................................................................................................................................... 52
Mono-en trisomiën ........................................................................................................................... 53
Aneuploïdie ................................................................................................................................... 53
Vorming mozaïeken....................................................................................................................... 53
Geslachtschromosale trisomiëen .................................................................................................. 53
Structurele chromosoomafwijkingen ................................................................................................ 54
Niet-gebalanceerde herschikking .................................................................................................. 54
Gebalanceerde herschikking ......................................................................................................... 54
LES 11: Overerving van gekoppelde genen ........................................................................................... 55
Gekoppelde genen ............................................................................................................................ 55
5
, Eerste proeven van Morgan .......................................................................................................... 55
Terugkruising ................................................................................................................................. 55
Crossing-over ..................................................................................................................................... 56
Chromosomenkaart....................................................................................................................... 56
Dubbele crossing-over ................................................................................................................... 56
Driepuntskruisingen ...................................................................................................................... 56
LOD-score (examenvraag) ................................................................................................................. 57
Recombinatiefequentie ................................................................................................................. 57
Hypotetische LOD-score ................................................................................................................ 57
HapMap project................................................................................................................................. 57
LES 12: Populatiegenetica ..................................................................................................................... 57
Bevolking ........................................................................................................................................... 57
Formule van Hardy-Weinberg ........................................................................................................... 57
Allelenfrequentie op een bepaald moment .................................................................................. 58
De genotypenfrequentie ............................................................................................................... 58
Besluit ............................................................................................................................................ 58
Voorwaarden ................................................................................................................................. 58
Consanguiniteit of bloedverwantschap............................................................................................. 58
Verwantschapscoëfficiënt r ........................................................................................................... 58
Inteeltcoëfficiënt F ........................................................................................................................ 58
Voorbeeld ...................................................................................................................................... 58
LES 13: Analysemethoden voor nucleïnezuren ..................................................................................... 59
DNA en RNA isolatie .......................................................................................................................... 59
DNA isolatie ................................................................................................................................... 59
mRNA isolatie ................................................................................................................................ 59
Elektroforese ..................................................................................................................................... 59
Gel-elektroforese........................................................................................................................... 59
Hybridisatie ....................................................................................................................................... 60
FISH ................................................................................................................................................ 60
DNA probes ................................................................................................................................... 60
Proces ............................................................................................................................................ 60
Methoden ...................................................................................................................................... 61
Southern Blotting .......................................................................................................................... 61
DNA sequencing ................................................................................................................................ 61
LES 14: Amplificatie van erfelijk materiaal ............................................................................................ 62
Wat is PCR?........................................................................................................................................ 62
6
, Principe .............................................................................................................................................. 62
RNA als target ................................................................................................................................ 62
PCR-cyclus.......................................................................................................................................... 62
Denaturatie.................................................................................................................................... 62
Annealing ....................................................................................................................................... 62
Elongatie ........................................................................................................................................ 62
Componenten .................................................................................................................................... 63
Primers .......................................................................................................................................... 63
dNTP’s ............................................................................................................................................ 63
Buffer ............................................................................................................................................. 63
Taq DNA-polymerase..................................................................................................................... 63
Optimalisatie en risico PCR................................................................................................................ 63
Voorkomen ‘False annealing’ ............................................................................................................ 64
Nested ‘primer’ PCR ...................................................................................................................... 64
‘Hot-start’ PCR ............................................................................................................................... 64
‘Touch-down’ PCR ......................................................................................................................... 64
‘Time release’ PCR ......................................................................................................................... 64
LES 15: PCR varianten ............................................................................................................................ 65
PCR-RFLP............................................................................................................................................ 65
RT-PCR: mRNA detectie ..................................................................................................................... 65
Multiplex PCR .................................................................................................................................... 65
Interrepeat PCR: DNA fingerprinting ............................................................................................. 65
Repeat-PCR ........................................................................................................................................ 66
Capillaire elektroforese ................................................................................................................. 66
Analyse cap elektroforese ............................................................................................................. 66
Kwantitatieve fluorescentie PCR/QF PCR .......................................................................................... 67
Allel-specifieke amplificatie............................................................................................................... 67
LES 16: Kwantitatieve PCR/ real time PCR............................................................................................. 68
Principe .............................................................................................................................................. 68
Klassieke kwantificatie PCR product ............................................................................................. 68
Geen agarose gel ........................................................................................................................... 68
Detectiemethoden ............................................................................................................................ 68
SYBR GREEN ................................................................................................................................... 68
Förster Resonance Energy Transfer .............................................................................................. 68
FRET-technologie ............................................................................................................................... 69
Hydrolyse probe ........................................................................................................................... 69
7
, Molecular beacons ........................................................................................................................ 69
Scorpions ....................................................................................................................................... 69
Dual-probes ................................................................................................................................... 69
Kwantificatie ...................................................................................................................................... 70
Dynamisch bereik .......................................................................................................................... 70
Smeltcurve analyse............................................................................................................................ 70
Afgeleide curve .............................................................................................................................. 70
LES 16a: Digital Droplet PCR .................................................................................................................. 71
Principe .............................................................................................................................................. 71
Verdeling ....................................................................................................................................... 71
Workflow ........................................................................................................................................... 71
Staalvoorbereiding ........................................................................................................................ 71
Vorming druppels .......................................................................................................................... 71
Verdeling target over druppels ..................................................................................................... 72
PCR................................................................................................................................................. 72
Hydrolyse probes........................................................................................................................... 72
Detectie ......................................................................................................................................... 72
Voordelen .......................................................................................................................................... 73
Hoge gevoeligheid ......................................................................................................................... 73
Belang concentratie....................................................................................................................... 73
Robuust en reproduceerbaar ........................................................................................................ 73
Poisson correctie ............................................................................................................................... 73
Toepassingen ..................................................................................................................................... 74
Niet invasieve opvolging kankerpatiënt ........................................................................................ 74
8
,Les 1: Erfelijk materiaal
DNA
Chromosomen
• Mens heeft 46 chromosomen of 23paar chromosomen
• Elke stuk chromosomen bevat genen => in totaal 25 000
• (Zoals getoond komt DNA enkel voor bij het delen van de cel)
• Bestaat uit 2 chromatinen verbonden bij de centromeer
• Telomeer: uiteinde chromatine
• DNA omheen histone-EW (positief geladen)
Karyogram vd mens
• 46 chromosomen: 2 set van 23 verschillende exemplaren
o 44 autosomen + 2 geslachtschromosomen
• Ordeningscriteria: obv de lengte van de chromosomen
o P: korte arm; Q lange arm
• Bandenpatroon
o Heterochromatine: donkere zone; inactief DNA
o Euchromatine: lichtere zone
o Bandnummers: vb 7q21
Bouwstenen
Fosfo-ester binding
Beta-glycosidische binding
Fosfo-anhydride binding
• Nucleotine:
9
, • 4 mogelijke base: adenine, guanine, cytosine, thymine
• Fosfo-diësterbinding
Structuur
• Altijd van 5’ naar 3’!
• Baseparen
o A en T: 2H-bruggen
o C en G: 3H-bruggen
• Secundaire structuur
o Rechtsdraaiende alfa-helix
o Bèta-vlakken
• Tertiaire structuur: nucleosomen
Humaan DNA
• 23 chromosomen (verschillende) = n = haploïde aantal
• 3 miljard basenparen
• 2% = genen → 25.000 eiwitten (ORFs/OpenReadingFrame)
• Genen: intronen/exonen (prokaryoten: continu)
• 98% = ‘junk’
Uniek DNA (50%)
• Genen
• Niet-gen sequenties met eenmalig voorkomen
• Pseudo-genen
Repetitief DNA (50%) => satelliet
• Micro-satelliet (STR) = Short Tandem Repeats
o vb. (CA)n of triplet repeats (2-10)
• Mini-satelliet (VNTR) = Variable Number of Tandem Repeats
o groter (10-60), minder aantal keren, 1 plaats
• Macro-satelliet = ‘elementen’
o groot (200-1500), vele plaatsen, 1 maal
o SINE = short interspresed nuclear elements (gemiddeld 300 bp), meest voorkomend
=Alu repeat
o LINE = Long interspresed nuclear elements (gemiddeld 800 bp), meest voorkomend
=LINE-1
Verandering op de genen (Rep. DNA)
• SNP = single nucleotide polymorfisme
o heel groot aandeel id bevolking, niet schadelijk
• Mutatie = 1-2% vd bevolking, meestal iets schadelijk
10
Inhoud
Les 1: Erfelijk materiaal ........................................................................................................................... 9
DNA...................................................................................................................................................... 9
Chromosomen ................................................................................................................................. 9
Karyogram vd mens ......................................................................................................................... 9
Bouwstenen..................................................................................................................................... 9
Structuur ........................................................................................................................................ 10
Humaan DNA ..................................................................................................................................... 10
Uniek DNA (50%) ........................................................................................................................... 10
Repetitief DNA (50%) => satelliet .................................................................................................. 10
Verandering op de genen (Rep. DNA) ........................................................................................... 10
Samenstelling ................................................................................................................................ 11
Satelliet DNA.................................................................................................................................. 11
Waar zit DNA ..................................................................................................................................... 12
Chromosomaal DNA ...................................................................................................................... 12
Extrachromosomaal DNA: circulair ............................................................................................... 12
Mitochondriaal DNA ...................................................................................................................... 12
Les 2: Van gen naar eiwit: transcriptie .................................................................................................. 13
Inleiding ............................................................................................................................................. 13
Soorten EW........................................................................................................................................ 13
Structurele EW .............................................................................................................................. 13
Transport-EW ................................................................................................................................ 14
Bewegings-EW ............................................................................................................................... 14
Reserve-EW ................................................................................................................................... 14
Signaal- en receptor-EW ................................................................................................................ 14
Gen-regulatorische EW ................................................................................................................. 14
Beschermende EW ........................................................................................................................ 14
Het dogma van Crick ......................................................................................................................... 14
Structuur van RNA ............................................................................................................................. 15
Soorten RNA .................................................................................................................................. 15
Verschil DNA en RNA ..................................................................................................................... 15
Eukaryote genen............................................................................................................................ 15
Polymerase II genen! ..................................................................................................................... 16
1
, Structuur van een gen ....................................................................................................................... 16
Transcriptie........................................................................................................................................ 17
Transcriptiefactoren ...................................................................................................................... 17
Structuur van mRNA .......................................................................................................................... 17
PTM 1 = post transcriptionele modificatie 1 ..................................................................................... 18
Cap structuur ................................................................................................................................. 18
Spliceosoom .................................................................................................................................. 18
Alternatieve splitsing ..................................................................................................................... 18
Les 3: De weg van gen naar EW 2 .......................................................................................................... 19
Genetische code ................................................................................................................................ 19
Translatie ........................................................................................................................................... 20
tRNA............................................................................................................................................... 20
Ribosomen ..................................................................................................................................... 20
Fasen van de translatie.................................................................................................................. 21
Groeiende polypeptideketen ........................................................................................................ 22
Transcriptie en translatie in prokaryoten en eukaryoten ............................................................. 22
PTM 2 = post translationele modificatie 2 ........................................................................................ 22
Gesecreteerde EW......................................................................................................................... 22
Glycosylatie ................................................................................................................................... 23
Fosforylatie .................................................................................................................................... 23
Acetylering..................................................................................................................................... 23
Hydroxylatie van proline ............................................................................................................... 24
Partiële proteolyse ........................................................................................................................ 24
Soorten proteasen ......................................................................................................................... 24
PTM 1 vs. PTM 2 ................................................................................................................................ 24
LES 4: Regulatie van genexpressie......................................................................................................... 25
Inleiding ............................................................................................................................................. 25
Regulatie van eiwitsynthese .............................................................................................................. 25
Voorbeelden EUK-genregulatie: .................................................................................................... 26
Epigenetica ........................................................................................................................................ 27
Mechanismen ................................................................................................................................ 28
DNA methylatie: ................................................................................................................................ 28
Overerving: .................................................................................................................................... 28
Genomische inprinting: ................................................................................................................. 28
Histonmodificatie .............................................................................................................................. 29
RNA interferentie .............................................................................................................................. 29
2
, Pathway ......................................................................................................................................... 30
Dicer: ............................................................................................................................................. 30
Sequentie specifieke mRNA degradatie: ....................................................................................... 30
miRNA: ........................................................................................................................................... 30
LES 5: Celdeling en overerving van het geslacht ................................................................................... 31
Inleiding ............................................................................................................................................. 31
De celcyclus ....................................................................................................................................... 31
G1: werkingsfase ........................................................................................................................... 31
S: synthese ..................................................................................................................................... 31
G2: laatste controle voor deling .................................................................................................... 32
M: delingsfase ............................................................................................................................... 32
Replicatie ........................................................................................................................................... 32
Replicatievork ................................................................................................................................ 32
Proof reading ................................................................................................................................. 33
Leading en lagging streng synthese............................................................................................... 33
Verlies van DNA ............................................................................................................................. 34
Werking telomerase ...................................................................................................................... 34
Soort deling ....................................................................................................................................... 34
Mitose............................................................................................................................................ 34
Meiose ........................................................................................................................................... 35
Herverdeling van het erfelijk materiaal............................................................................................. 35
Crossing over ................................................................................................................................. 35
Segregatie ...................................................................................................................................... 36
De voortplantingscellen/ gameten.................................................................................................... 36
De man: ......................................................................................................................................... 36
De vrouw: ...................................................................................................................................... 37
Geslachtsbepaling bij bevruchting ................................................................................................ 37
Sexratio .......................................................................................................................................... 37
Y-chromosoom .............................................................................................................................. 38
X-chromosoom .............................................................................................................................. 38
LES 6: Mutaties ...................................................................................................................................... 39
Puntmutaties ..................................................................................................................................... 39
Sikkelcelanemie: ............................................................................................................................ 39
Voorkomen mutaties..................................................................................................................... 40
Micro-deleties en –inserties .............................................................................................................. 40
Trinucleotide repeat amplificaties (dynamische mutaties)............................................................... 40
3
, Mutaties die de transcriptie of mRNA processing verstoren ............................................................ 41
Lengtemutaties.................................................................................................................................. 42
Soorten .......................................................................................................................................... 42
Ongelijke recombinatie ................................................................................................................. 42
Translocaties.................................................................................................................................. 42
Transpositie ................................................................................................................................... 42
Oorzaken en gevolgen mutaties........................................................................................................ 43
Oorzaken spontane mutaties ........................................................................................................ 43
Externe invloeden door mutagenen.............................................................................................. 43
Gevolgen mutaties ........................................................................................................................ 43
LES 7: Monogene overerving ................................................................................................................. 44
Inleiding ............................................................................................................................................. 44
Kruiswetten van Mendel ............................................................................................................... 44
Basisbegrippen .................................................................................................................................. 45
Genotype en fenotype: ................................................................................................................. 45
Variabiliteit .................................................................................................................................... 45
Genotype naar fenotype ............................................................................................................... 45
Ontstaan ziekten ............................................................................................................................... 45
Recessieve ziekten ......................................................................................................................... 45
Dominante ziekten ........................................................................................................................ 45
Stamboom ......................................................................................................................................... 46
Autosomale overerving ..................................................................................................................... 46
Recessieve aandoeningen ............................................................................................................. 46
Dominante aandoeningen ............................................................................................................. 46
Partiële dominantie ....................................................................................................................... 47
Codominantie bij ABO bloedgroep................................................................................................ 47
LES 8: Geslachtsgebonden overerving .................................................................................................. 47
Geslachtsgebonden kenmerken ........................................................................................................ 47
X-gebonden recessieve aandoeningen ............................................................................................. 47
Stamboom ..................................................................................................................................... 47
Hemofilie/ bloederziekte............................................................................................................... 48
Duchenne(DMD) ............................................................................................................................ 48
Becker(BMD) ................................................................................................................................. 48
Fragiele X-syndroom...................................................................................................................... 48
Daltonisme .................................................................................................................................... 48
Syndroom van Lesch-Nyhan .......................................................................................................... 48
4
, Variaties in G6PDH ........................................................................................................................ 48
X-gebonden dominante aandoeningen............................................................................................. 48
Stamboom ..................................................................................................................................... 49
Syndroom van Rett (RTT)............................................................................................................... 49
Het Y-chromosoom............................................................................................................................ 49
Overerving ..................................................................................................................................... 49
Stamboom ..................................................................................................................................... 49
Mitochondriale overerving ................................................................................................................ 49
Kenmerken .................................................................................................................................... 49
Stamboom ..................................................................................................................................... 49
LES 9 : Multifactoriële overerving ......................................................................................................... 50
Inleiding ............................................................................................................................................. 50
Beïnvloeding door meerdere genen.................................................................................................. 50
Additieve polygenie ....................................................................................................................... 50
Drempel polygenie ........................................................................................................................ 50
Epistase en hypostase ................................................................................................................... 50
Beïnvloeding vanuit de omgeving ..................................................................................................... 51
Complicerende processen ................................................................................................................. 51
Locus heterogeniteit...................................................................................................................... 51
Pleiotropie ..................................................................................................................................... 51
Penetrantie en expressiviteit ........................................................................................................ 51
Mozaïken ....................................................................................................................................... 51
Genetische anticipatie ................................................................................................................... 52
Epi genetische invloeden( zie Epigenetica) ................................................................................... 52
LES 10: chromosomale afwijkingen ....................................................................................................... 52
Inleiding ............................................................................................................................................. 52
Polyploïdie ......................................................................................................................................... 52
Mono-en trisomiën ........................................................................................................................... 53
Aneuploïdie ................................................................................................................................... 53
Vorming mozaïeken....................................................................................................................... 53
Geslachtschromosale trisomiëen .................................................................................................. 53
Structurele chromosoomafwijkingen ................................................................................................ 54
Niet-gebalanceerde herschikking .................................................................................................. 54
Gebalanceerde herschikking ......................................................................................................... 54
LES 11: Overerving van gekoppelde genen ........................................................................................... 55
Gekoppelde genen ............................................................................................................................ 55
5
, Eerste proeven van Morgan .......................................................................................................... 55
Terugkruising ................................................................................................................................. 55
Crossing-over ..................................................................................................................................... 56
Chromosomenkaart....................................................................................................................... 56
Dubbele crossing-over ................................................................................................................... 56
Driepuntskruisingen ...................................................................................................................... 56
LOD-score (examenvraag) ................................................................................................................. 57
Recombinatiefequentie ................................................................................................................. 57
Hypotetische LOD-score ................................................................................................................ 57
HapMap project................................................................................................................................. 57
LES 12: Populatiegenetica ..................................................................................................................... 57
Bevolking ........................................................................................................................................... 57
Formule van Hardy-Weinberg ........................................................................................................... 57
Allelenfrequentie op een bepaald moment .................................................................................. 58
De genotypenfrequentie ............................................................................................................... 58
Besluit ............................................................................................................................................ 58
Voorwaarden ................................................................................................................................. 58
Consanguiniteit of bloedverwantschap............................................................................................. 58
Verwantschapscoëfficiënt r ........................................................................................................... 58
Inteeltcoëfficiënt F ........................................................................................................................ 58
Voorbeeld ...................................................................................................................................... 58
LES 13: Analysemethoden voor nucleïnezuren ..................................................................................... 59
DNA en RNA isolatie .......................................................................................................................... 59
DNA isolatie ................................................................................................................................... 59
mRNA isolatie ................................................................................................................................ 59
Elektroforese ..................................................................................................................................... 59
Gel-elektroforese........................................................................................................................... 59
Hybridisatie ....................................................................................................................................... 60
FISH ................................................................................................................................................ 60
DNA probes ................................................................................................................................... 60
Proces ............................................................................................................................................ 60
Methoden ...................................................................................................................................... 61
Southern Blotting .......................................................................................................................... 61
DNA sequencing ................................................................................................................................ 61
LES 14: Amplificatie van erfelijk materiaal ............................................................................................ 62
Wat is PCR?........................................................................................................................................ 62
6
, Principe .............................................................................................................................................. 62
RNA als target ................................................................................................................................ 62
PCR-cyclus.......................................................................................................................................... 62
Denaturatie.................................................................................................................................... 62
Annealing ....................................................................................................................................... 62
Elongatie ........................................................................................................................................ 62
Componenten .................................................................................................................................... 63
Primers .......................................................................................................................................... 63
dNTP’s ............................................................................................................................................ 63
Buffer ............................................................................................................................................. 63
Taq DNA-polymerase..................................................................................................................... 63
Optimalisatie en risico PCR................................................................................................................ 63
Voorkomen ‘False annealing’ ............................................................................................................ 64
Nested ‘primer’ PCR ...................................................................................................................... 64
‘Hot-start’ PCR ............................................................................................................................... 64
‘Touch-down’ PCR ......................................................................................................................... 64
‘Time release’ PCR ......................................................................................................................... 64
LES 15: PCR varianten ............................................................................................................................ 65
PCR-RFLP............................................................................................................................................ 65
RT-PCR: mRNA detectie ..................................................................................................................... 65
Multiplex PCR .................................................................................................................................... 65
Interrepeat PCR: DNA fingerprinting ............................................................................................. 65
Repeat-PCR ........................................................................................................................................ 66
Capillaire elektroforese ................................................................................................................. 66
Analyse cap elektroforese ............................................................................................................. 66
Kwantitatieve fluorescentie PCR/QF PCR .......................................................................................... 67
Allel-specifieke amplificatie............................................................................................................... 67
LES 16: Kwantitatieve PCR/ real time PCR............................................................................................. 68
Principe .............................................................................................................................................. 68
Klassieke kwantificatie PCR product ............................................................................................. 68
Geen agarose gel ........................................................................................................................... 68
Detectiemethoden ............................................................................................................................ 68
SYBR GREEN ................................................................................................................................... 68
Förster Resonance Energy Transfer .............................................................................................. 68
FRET-technologie ............................................................................................................................... 69
Hydrolyse probe ........................................................................................................................... 69
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, Molecular beacons ........................................................................................................................ 69
Scorpions ....................................................................................................................................... 69
Dual-probes ................................................................................................................................... 69
Kwantificatie ...................................................................................................................................... 70
Dynamisch bereik .......................................................................................................................... 70
Smeltcurve analyse............................................................................................................................ 70
Afgeleide curve .............................................................................................................................. 70
LES 16a: Digital Droplet PCR .................................................................................................................. 71
Principe .............................................................................................................................................. 71
Verdeling ....................................................................................................................................... 71
Workflow ........................................................................................................................................... 71
Staalvoorbereiding ........................................................................................................................ 71
Vorming druppels .......................................................................................................................... 71
Verdeling target over druppels ..................................................................................................... 72
PCR................................................................................................................................................. 72
Hydrolyse probes........................................................................................................................... 72
Detectie ......................................................................................................................................... 72
Voordelen .......................................................................................................................................... 73
Hoge gevoeligheid ......................................................................................................................... 73
Belang concentratie....................................................................................................................... 73
Robuust en reproduceerbaar ........................................................................................................ 73
Poisson correctie ............................................................................................................................... 73
Toepassingen ..................................................................................................................................... 74
Niet invasieve opvolging kankerpatiënt ........................................................................................ 74
8
,Les 1: Erfelijk materiaal
DNA
Chromosomen
• Mens heeft 46 chromosomen of 23paar chromosomen
• Elke stuk chromosomen bevat genen => in totaal 25 000
• (Zoals getoond komt DNA enkel voor bij het delen van de cel)
• Bestaat uit 2 chromatinen verbonden bij de centromeer
• Telomeer: uiteinde chromatine
• DNA omheen histone-EW (positief geladen)
Karyogram vd mens
• 46 chromosomen: 2 set van 23 verschillende exemplaren
o 44 autosomen + 2 geslachtschromosomen
• Ordeningscriteria: obv de lengte van de chromosomen
o P: korte arm; Q lange arm
• Bandenpatroon
o Heterochromatine: donkere zone; inactief DNA
o Euchromatine: lichtere zone
o Bandnummers: vb 7q21
Bouwstenen
Fosfo-ester binding
Beta-glycosidische binding
Fosfo-anhydride binding
• Nucleotine:
9
, • 4 mogelijke base: adenine, guanine, cytosine, thymine
• Fosfo-diësterbinding
Structuur
• Altijd van 5’ naar 3’!
• Baseparen
o A en T: 2H-bruggen
o C en G: 3H-bruggen
• Secundaire structuur
o Rechtsdraaiende alfa-helix
o Bèta-vlakken
• Tertiaire structuur: nucleosomen
Humaan DNA
• 23 chromosomen (verschillende) = n = haploïde aantal
• 3 miljard basenparen
• 2% = genen → 25.000 eiwitten (ORFs/OpenReadingFrame)
• Genen: intronen/exonen (prokaryoten: continu)
• 98% = ‘junk’
Uniek DNA (50%)
• Genen
• Niet-gen sequenties met eenmalig voorkomen
• Pseudo-genen
Repetitief DNA (50%) => satelliet
• Micro-satelliet (STR) = Short Tandem Repeats
o vb. (CA)n of triplet repeats (2-10)
• Mini-satelliet (VNTR) = Variable Number of Tandem Repeats
o groter (10-60), minder aantal keren, 1 plaats
• Macro-satelliet = ‘elementen’
o groot (200-1500), vele plaatsen, 1 maal
o SINE = short interspresed nuclear elements (gemiddeld 300 bp), meest voorkomend
=Alu repeat
o LINE = Long interspresed nuclear elements (gemiddeld 800 bp), meest voorkomend
=LINE-1
Verandering op de genen (Rep. DNA)
• SNP = single nucleotide polymorfisme
o heel groot aandeel id bevolking, niet schadelijk
• Mutatie = 1-2% vd bevolking, meestal iets schadelijk
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