PCR is involved in many applications, such as DNA sequencing, cloning, DNA engineering, clinical diagnosis, and gene expression detection.  Formerly, scientists used the intensity of a fluorescent light from EB or used a densitometry device to scan gel images in order to estimate the expression level of differently- targeted sequences.  As a result, there were drawbacks with these methods to measure expression levels accurately.
During PCR, the accumulation of end products has three stages:

• Exponential stage: The end product doubles after each cycle.  The reaction is precise and specific.
• Linear stage: The reaction becomes slower because the consumption of reaction components, such as primers and dNTPs, start to degrade.
• Plateau stage:  The reaction has stopped.  Partial end product degradation is ongoing.

The end point analysis, which is based on gel electrophoresis, is analyzed at a signal during the plateau stage, which will have very high variability.  The following graph demonstrates that a sample replicate can result in a different intensity.

As stated above, quantity determination at exponential stage is the most accurate, and real-time PCR is the method that can capture such a change.
The real-time PCR technology is comprised of three major components:

• An involvement of an increased amount of fluorescent molecules during PCR cycles, thus the DNA amplification can be monitored in real time. 
• A real-time PCR instrument is, simply, a thermal cycler with a high resolution camera box on top .
• An instrument software that will drive the instrument through thermal condition, collect fluorescent intensity from PCR reaction, and convert such readings to quantitative data.

There two chemistries for real-time PCR:

• Double-stranded DNA binding dye detection:

The dye molecule will only bind to double-stranded DNA, such as a PCR product, and it will only emit fluorescent light when it binds to double-stranded DNA; the fluorescent intensity will increase when more double-stranded DNAs are replicated during thePCR cycle.  The dye often used from this chemistry is SYBR green.  Because the dye can bind to any double-stranded DNA, the specificity is its disadvantage.

• Fluorescent reporter probe detection:

Besides a pair of PCR primers, a probe is designed in between that its sequence is complementary to the target DNA.  On one end of the probe, it has a reporter dye molecule, and on the other end, it has a quencher molecule.  Because of the frequency resonance energy transfer (FRET) and the close distance between the reporter dye and the quencher, the fluorescent energy from the reporter dye will be absorbed by  the quencher.  During PCR reaction, the spatial relationship between the reporter dye and the quencher changes and fluorescent light  emitted from the dye can be detected by real-time PCR instrument.  This chemistry, because of the probe design, is more specific than SYBR green based detection.  In addition, with different reporter dye labeling, detection of multiple sequences in one reaction tube (multiplex reaction) becomes possible.