Real Time PCR Flashcards
What are real time PCR chemistries?
these are the methods used to create and detect fluorescence upon DNA amplification.
What are the 2 major chemistries?
SYBR green
TaqMan (5’ nuclease) probe
What is SYBR green?
It is a fluorogenic dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to ds-DNA.
When is SYBR used over TaqMan?
- When high specificity is not needed or required
- General screening of targets before moving to probe-based assays
- For high resolution melting curve analysis of SNPs
- More flexibility since it doesn’t require designing a separate probe
What are the limitations of SYBR green?
- It binds to any dsDNA so not specific for desired amplified sequence.
- Primer dimers will also generate signals from sybr green
- Requires melting/dissociation curve analysis after PCR
What is the use of the MC?
Distinguish between primer dimers and specific product
How is the MC initiated?
The machine is programmed to do a MC in which the temperature is raised by a fraction of a degree and the change in fluorescence is measured. At MP, the two strands of DNA separate and fluorescence rapidly decreases.
The melting point of a dsDNA depends on what?
Its base composition and length
All PCR products from a particular primer pair should all have the same melting point unless
there is contamination, mispriming, primer dimer artifacts or some other problem.
What is the quality control measure specific to SYBR green which ensures non-specific amplification is not issue?
Running a melt curve
Small peaks with lower Tm usually indicate
Primer dimers
this chemistry takes advantage of the natural 5’-3’ exonuclease activity of Taq polymerase when it is building a new strand
Taqman 5’ nuclease probe
What is the mechanism of 5’ nuclease probes as chemistries?
As Taq polymerase extends the new strand during PCR, it has the ability to cleave any nucleotide in its way. With 5’ nuclease probe, it is designed to anneal on the template between the forward and reverse primers. Cleavage of part of this probe by Taq polymerase is what produces fluorescence during extension.
Green emission of the probe is suppressed by the
quencher
When can the probe emit green light?
When quencher is separated from it
FRET stands for
Fluorescent resonant energy transfer
What does Taqman probe use to inhibit fluorescence till PCR amplification?
FRET
Why is Taqman called a hydrolysis probe?
Because it is the cleavage from tan polymerase that cause fluorescence to occur
What is the difference between FRET usage in hydrolysis and hybridization probes
Hydrolysis probes use FRET to inhibit fluorescence till amplification, but hybridization probes use FRET to enhance fluorescence.
List two other mixed mechanism probes
Molecular beacons
Scorpion probes
What are hybridization probes used for?
Light cycler technology
What modifications have been done on Taqman to improve it?
Made shorter for it to be more specific.
Minor groove binder (MGB) moiety added
Non fluorescent quencher
Sensitive to single-base matches
