Quantitative PCR Flashcards
In what way are PCR reactions not quantitative?
It isn’t quantitative because it doesn’t quantify the absolute amount of transcripts, rather, it quantifies them in relation to one another.
How can you make a PCR quantitative?
By adding a template which has a known concentration that utilizes the same primers as the transcript of interest. This way, you’ll know the degree to which it’s been amplified.
This is known as transcript quantification by competative RT-PCR.
What’s the differrence between PCR and qPCR (Real time PCR)?
A PCR amplfiies the DNA for each cycle. A qPCR uses a fluorescent dye to quantify the light expression after each cycle.
How does SYBRgreen work?
It’s an intercalating dye which binds non-specifically in freshly polymerised sequences.
How does taqMan probes work?
TaqMan probes are oligos with one fluorophore per end. The probes bind to ssDNA at a specific sequence. When the DNApol polymerases the complementary strand, the TaqMan probe gets degraded by the polymerase’s exonucleic activity, releasing one of the fluoropores (which was previously quenched by the other one).
What’s the difference between olecular beacons and taqman probes?
TaqMan probes get degraded during the cycles. Molecular beacons remain intact and bind to the target in every cycle.
Are there any cons of SYBRgreen?
- The fluorescence is non-specific, it doesn’t matter what is bound, the fluorophore fluoresces never the less.
- Primer dimers get amplified. It’s important to verify RT-qPCR results for this reason.
When studying splicing variants, would you use taqMan or SYBRgreen?
TaqMan because you can map it to an exon which is only present in a certain splice variant.
Define ‘Ct value*.
Ct-value is the number of cycles required for the fluorimeter to segregate the fluorescence of a sample from background fluorescence.
