Via de theorielessen en de practica is het de bedoeling om aan de toekomstige labotechnologen op het vlak van de moleculaire genetica zowel een ruime theoretische als praktische bagage mee te geven. Moleculaire detectietechnieken worden dagelijks gebruikt in allerhande onderzoekslabo’s en mogen d...
SAMENVATTING MOLECULAIRE BIOLOGIE II
Inhoud
PCR of polymerase kettingreactie.............................................................................................................................4
Principe van de techniek.......................................................................................................................................4
Componenten noodzakelijk voor PCR – reactie in vivo....................................................................................4
Repetitieve cyclus van PCR – techniek..............................................................................................................5
Voorbeeld PCR – reactie (3 cyclussen)..................................................................................................................6
PCR – cyclus 1....................................................................................................................................................6
PCR – cyclus 2....................................................................................................................................................7
PCR – cylcus 3....................................................................................................................................................7
Reactievoorwaarden voor PCR..............................................................................................................................8
Keuze van de primers (aan wat moeten primers voldoen)...............................................................................9
Vuistregels voor PCR (Oefening examen)............................................................................................................9
Maken van een mastermix................................................................................................................................9
Toepassingen van PCR – technologie......................................................................................................................10
Opsporen van specifiek DNA – fragmentje.........................................................................................................10
Aantonen van puntmutaties via CAPS of PCR – RFLP.........................................................................................11
Voorbeeld: Sikkelcelanemie............................................................................................................................12
Wildtype wIJZIGT IN t......................................................................................................................................12
Aantonen van deletie via PCR.........................................................................................................................14
Aantonen van insertie via PCR........................................................................................................................15
DNA fingerprinting..............................................................................................................................................16
STR en microsatelliet.......................................................................................................................................16
Voorwaarden voor een multiplex PCR............................................................................................................16
Maken van genetische vingerafdrukken.............................................................................................................17
Fragment analyse via capillaire elektroforese................................................................................................17
Toepassing van DNA – fingerprinting..................................................................................................................18
Reverse Transcriptase PCR......................................................................................................................................20
Principe van deze techniek..................................................................................................................................21
HIV en reverse transcriptase...............................................................................................................................21
Productie van Copy DNA (cDNA) en PCR.............................................................................................................23
1. Synthese van cDNA m.b.v. OligodT (Kunnen tekenen)...............................................................................23
2. Reverse Primer (Kunnen tekenen)..............................................................................................................24
Toepassingen reverse Transcriptase PCR............................................................................................................24
A) Genexpressie...............................................................................................................................................24
1
, B) Voorkomen van alternatieve splicingsproducten.......................................................................................24
C) Opsporen van levende micro – organismen...............................................................................................26
D) Opsporen van Micro – organismen met RNA als genoom.........................................................................26
Real time PCR..........................................................................................................................................................26
Principe van Real time PCR.................................................................................................................................26
Verschil tussen klassieke PCR en Real time PCR.............................................................................................26
Waarom real time – PCR.................................................................................................................................26
Voordelen van Real – time PCR t.o.v. klassieke PCR.......................................................................................26
Fasen in een S vormige PCR-amplificatie curve..............................................................................................27
Real time PCR met SYBR green............................................................................................................................28
Real time PCR met een probe.............................................................................................................................29
Taqman – probe..............................................................................................................................................29
Delta Rn en Cycle treshhold (Ct).........................................................................................................................30
Bekomen van Rn en Delta Rn..........................................................................................................................30
Recombinante DNA technologie.............................................................................................................................30
restrictie – enzymen............................................................................................................................................30
Naamgeving restrictie – enzymen...................................................................................................................30
Voorbeelden restrictie – enzymen..................................................................................................................31
Plasmiden/Vectoren en waardcellen..................................................................................................................32
Plasmiden........................................................................................................................................................32
Uitgangsmateriaal voor klonering.......................................................................................................................32
Welk DNA wordt in de vectoren gekloneerd..................................................................................................32
Klonering van DNA – 3 essentiële stappen.........................................................................................................33
Het maken van een recombinant plasmide........................................................................................................34
Modificatie van DNA – eindjes........................................................................................................................34
Defosforlyatie van eindstandige 5’ fosfaatgroep................................................................................................35
Ligatie van de eindjes..........................................................................................................................................36
Bacteriële transformatie.....................................................................................................................................37
Selecteren en oppikken van de goede kolonies..................................................................................................37
Via PCR de richting van het insert bepalen.....................................................................................................38
Sequentieanalyse....................................................................................................................................................38
Dideoxymethode van Sanger..............................................................................................................................38
Algemeen principe..........................................................................................................................................38
Polyacrylamide gelektroforese onder denaturerende condities....................................................................40
Aflezing van een sequentiegel.............................................................................................................................41
Manueel – Autoradiogram..............................................................................................................................41
Automatische sequentie – analyse.................................................................................................................41
2
, Cycle sequenering...............................................................................................................................................42
Hoe werkt cycle sequenering..........................................................................................................................42
Hoe PCR – fragment sequeneren....................................................................................................................43
Illumina sequencing methode.............................................................................................................................44
Principe............................................................................................................................................................44
Handelingen voor het sequeneren van een genoom via Illumina methode (Examen –kunnen opsommen) 44
Reversibel nucleotide (kunnen uitleggen).......................................................................................................47
Mutagenese van DNA..............................................................................................................................................48
PRINCIPE VAN MUTAGENESE..............................................................................................................................48
Doel mutagenese............................................................................................................................................48
Gerichte mutagenese via quick change site directed mutagenesis....................................................................48
Werkwijze gerichte mutagenese.....................................................................................................................49
Toepassingen quick change mutagenese........................................................................................................50
Deletie aanbrengen.........................................................................................................................................51
Moleculaire biologie II – practicum.........................................................................................................................52
Knippen van DNA met restrictie – enzymen.......................................................................................................52
Principe............................................................................................................................................................52
Opstellen van een Digestschema....................................................................................................................52
Gecombineerde digesten....................................................................................................................................53
Opstellen van een digestschema (Van buiten leren)......................................................................................53
PCR in praktijk.....................................................................................................................................................57
Remmende factoren van de PCR....................................................................................................................57
PCR – reactiecomponenten.............................................................................................................................58
Samenvattende vragen - PCR in praktijk.............................................................................................................59
Begrippenlijst Moleculaire Biologie II.....................................................................................................................61
PCR OF POLYMERASE KETTINGREACTIE
3
, PRINCIPE VAN DE TECHNIEK
Door een PCR – reactie op DNA – fragmenten uit te voeren kan er een grote hoeveelheid van het
gewenste stukje DNA gevormd worden
Het is een cyclisch (gebeurt bij temperatuurverloop) in vitro DNA amplificatieproces waarbij het
fenomeen in vivo replicatie wordt nagebootst
Replicatie van DNA in vivo
COMPONENTEN NOODZAKELIJK VOOR PCR – REACTIE IN VIVO
DNA – template
Twee primers (Forward en Reverse primer)
dNTP’ s (Nucleoside trifosfaat)
DNA – polymerase Eppendorf
Geschikte buffer (magnesiumchloride of DMSO)
Thermocycler
TEMPLATE
Voorwaarde startmateriaal:
o Het moet de sequentie bevatten die geamplificeerd dient te worden
Volledige genoom niet nodig
Kan zowel gebruik maken van:
o Chromosomaal
o Extra chromosomaal DNA (plasmide DNA)
o Ook viraal DNA
REPETITIEVE CYCLUS VAN PCR – TECHNIEK
4
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