Nucleic Acid Amplification - Target Amplification
9 important questions on Nucleic Acid Amplification - Target Amplification
Wat is contaminatie controle in PCR mbv psoralen?
Wat is real-time of kwantitatieve PCR (RT-/qPCR)?
- Er wordt een probe toegevoegd of kleuring om een fluorescent signaal te genereren
- detectie van signaal in real time zorgt voor kwantificering van startmateriaal
- gespecialiseerde thermal cyclers met fluorescente detectie-systemen
Wat is de threshold cycle (Ct) in qPCR?
De threshold (drempel) is het punt van detectie, dit is meestal 10x de SD van de achtergrond fluorescentie.
In qPCR is de template input omgekeerd gecorreleerd aan de drempelcyclus. (Log-functie)
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Wat zijn qPCR detectiesystemen?
- DNA-specifieke kleuringen zoals SYBRGreen
- Hybridisatie probes:
- Splitsing gebaseerd (TaqMan)
- verplaatsbaar (Molecular Beacons, FRET)
Hoe werkt detectie van qPCR met SYBRGreen?
- Bindt kleine groef van dubbelstrengs DNA.
- Postamplificatie smeltkromme: amplicons hebben karakteristieke smelttemperaturen.
Hoe werkt qPCR detectie met TaqMan probe?
- De TaqMan probe bevat een fluorescente kleurstof (reporter) aan het 5'-einde en een quencher aan het 3'-einde.
- Tijdens de verlengingsfase komt de polymerase de TaqMan probe tegen en klieft het de probe met zijn 5'-3' exonuclease-activiteit, waardoor de fluorescente reporter van de quencher wordt gescheiden.
- Na de probe-afbraakcyclus wordt de fluorescente reporter vrijgegeven en geactiveerd, waardoor het fluorescentiesignaal wordt gedetecteerd.
Hoe werkt qPCR detectie met molecular beacons?
Molecular beacons bestaan uit drie delen: een sequentie complementair aan het target-DNA-fragment (bindingssequentie), een fluorescerende kleurstof (reporter) aan het ene uiteinde en een quencher aan het andere uiteinde. Wanneer de molecular beacon in zijn haarspelcstructuur is gevouwen, is de fluorescentie van de reporter gedempt door de nabijheid van de quencher.
- Bij binding tijdens de verlengingsfase wordt de haarspeld geopend en de reporter van de quencher gescheiden waardoor de reporter wordt vrijgegeven en geactiveerd.
Hoe werkt qPCR detectie met FRET probes?
Wat is microbial load testing?
Detecteer actieve infectie door lading te verhogen.
- Drempelcyclus patiëntmonster bepalen
- Bacteriële lading patiëntmonster bepalen
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