Detectors/ Fluorescence Markers used in Real Time PCR
Real time PCR employs detector for the monitoring of the progress of reaction (amplification of DNA). Detectors used can be various fluorescence markers. Among them most common and important ones includes:
- Taqman Probe
- SYBR Green
Taqman assay is a widely used RT-PCR assay because of its sensitivity and specificity. It is based on 5′–3′ exonuclease activity of Taq DNA polymerase. TaqMan probe is a sequence-specific oligonucleotide with a reporter fluorescent dye at 5′ end and a quencher dye at 3′ end.
It is a hydrolysis probe which bear a reporter dye, usually fluorescein (FAM) at its 5’ end and a quencher tetramethylrhodamine (TAMRA), attached to the 3’ end of the oligonucleotide.
Under normal conditions, probe remain coiled on itself bringing the fluorescence dye near the quencher, which inhibits or quenches of fluorescent signal of the dye. i.e. When the probe is not hydrolyzed by Taq DNA polymerase, reporter dye emitted fluorescent light which is absorbed by quencher dye because of fluorescent resonance energy transfer (FRET).
The oligonucleotide of the Taq polymerase has a homologous region with the target gene and thus when the target sequence is present in the mixture, it bind with the sample DNA.
As Taq polymerase start to synthesize new DNA strand in the extension stage, it causes degradation of the probe by 5’ end nuclease activity and the fluorescein is separated from the quencher as a result of which fluorescence signal is generated. i.e.
When probe is hydrolyzed by Taq DNA polymerase, the 5′ reporter dye is separated from quencher dye, the quenching effect is gone, and fluorescent light emitted by reporter dye forms fluorescent signal.
The fluorescent signal from 5′ reporter dye is detected by RT-PCR instrument. The released 5′ reporter dye signal is proportional to the amount of PCR products.
As this procedure continues, in each cycle the number of signal molecule increases, causing the increase in fluorescence which is positively related with the amplification of the target.
- Increased Sensitivity.
- It can detect multiple genes in same well i.e. Multiplexing.
- Used for most accurate quantification of PCR product accumulation.
- Realtively expensive than other detection methods.
Nonspecific fluorescent dye SYBR works on principle that when SYBR dyes bind to double-stranded DNA, their fluorescence increases by 20–100-fold. As the amount of double-stranded DNA increases during PCR process, the SYBR fluorescent signal increases correspondingly. SYBR can bind any double-stranded DNA, even primer dimers or concatamer. So it is critical to optimize PCR reactions to amplify the target amplicon only.
This is a dye that emits prominent fluorescent signal when it binds at the minor groove of DNA, nonspecifically.
SYBR Green is more preferred as it can provide information about each cycle of amplification as well as about the melting temperature which is not possible in Taqman Probe.
Other fluorescent dyes like Ethidium Bromide or Acridine Orange can also be used but SYBR Green is better used for its higher signal intensity.
- Fluorescent labels probes are not required.
- Low cost i.e. Cost effective.
- Less specificity as compared to Taqman Probe.
- Multiplexing is not possible.