Summary
Moleculaire diagnostiek samenvatting
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- Institution
visuele samenvatting die alles samenvat uit de cursus van Moleculaire Diagnostiek
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MOLECULAIRE DIAGNOSTIEKqPCR
QPCR VS CONVENTIONELE PCR
qPCR Conventionele PCR
Kwantitatief Niet kwantitatief
Tijdens de reactie gemeten End point meting
Post PCR geen analyses meer doen Post PCR wel analyses doen contaminatie
contaminatie vermijden
Fluorochromen (meerdere tegelijk) Fragmentlengtes bepalen
ALGEMEEN PRINCIPE
DETECTIEMETHODE
Direct Indirect
Intercallerende kleurstoffen Hydrolyse probe (TaqMan)
Vb. SybrGreen
5’3’ exonucleaseactiviteit
1
, Molecular beacon probe
Wordt gebruikt bij GENOTYPERING
- Opsporen SNV
- Enkel binden bij 100%
complementariteit
- 2 verschillende: wild type en mutant
type
MULTPLEX-PCR
Verschillende fluorochromen toevoegen probleem: SPECTRALE OVERLAP
Oplossing: fluorochromen zoveel mogelijk scheiden obv emissie-eigenschappen en PCR-toestel
kalibreren
2
, DIGITAL DROPLET PCR
PCR-inhibitoren hebben minder effect op kwantificatie (verdund)
Target in lage hoeveelheid aanwezig
Diagnostiek:
Mutatie in minderheid van de cellen (tumor)
Circulating free-cell DNA
CNV
DETECTIE & KWANTIFICERING
Efficiëntie is afhankelijk van
- Temperatuur
- Primers
- Pipetteren
- PCR-inhibitoren (ethanol, heparine)
Kan berekend worden via een standaardcurve
of via de formule: aanvaardbaar tussen 90-110%
a= -3,32, dan is E= 100%
Kwantificeren= meten van de hoeveelheid fluorescentie in de reactie
Startmateriaal kan je via
- Externe standaard (vb. plasmide) = absolute kwantificatie
o Berekenen via ijklijn/standaardcurve
- Interne standaard berkenen = relatieve kwantificatie
o Berekenen door 2∆Cq
o Nut: data normaliseren, kwaliteit monster nagaan
2 methoden om genexpressie vast te stellen
Livak methode
3
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