Quantitative PCR (Real-time PCR) is a molecular biology technique for the amplification of targeted DNA molecules in real-time. Real-time PCR is a recently developed technique by Higuchi in 1993. This procedure uses a modified thermal cycler with UV Detector and Charged couple device (CCD) camera. The procedure measures the amount of amplicon produced in each cycle. Amplification and detection take place in a single tube and eliminates post PCR manipulations.
There are several stages to the PCR reaction.
In the Exponential stage, the PCR reagents are abundant and amplicons are doubled. In the linear stage, the reagents start to run off and PCR reactions slow down. In the plateau stage, the reagents are depleted and PCR reactions stop down.
- Nonspecific sybrGreen detection method
- Taqman probe
- Molecular beacons
Nonspecific sybr Green detection method
Syber Green binds to the minor groove of double-stranded DNA. Fluorescence increases with the concentration of double-stranded DNA.
Taqman Probe Detection method
Taqman probe is an oligonucleotide probe consists of reporter and quencher dye. When the reporter is excited by the light it transfers the energy to the quencher and prevents reporter dye to emit light. The cleavage of the probe takes place during the extension of the PCR reaction. When the light excites the reporter dye, the reporter dye can now emit light since the quencher dye is far to block the emission. Fluorescence increases as the quenched probe is digested.
Fluorescence increases as the quenched probe hybridize to the DNA template. Beacons consist of ssDNA with an internal complementary sequence that keeps reporter and quencher dyes very close. During the denaturation, beacons anneal to the template separating both dyes and fluorescence increases with the concentration of DNA.
Primers are labeled with the isomeric base and the fluor. The complementary base contains a quencher. The more the ds DNA produced, the amount of fluorescence decreases.
Quantitation is not needed after real-time PCR. Quantitative PCR is very significant in the diagnosis of infectious diseases, detection of human and plant pathogens. Reagents are expensive in the real-time PCR and improper pipetting of standards can cause large errors in precision.