We all know about real time PCR but, with so much information available on this technique and with so many different chemistries, I thought it would be a good idea to compile all that information and write a blog on it.
As the name suggests real time PCR is a type of PCR (polymerase chain reaction) that allows detection of amplified DNA in real time. A normal PCR requires some post -PCR procedures to be performed for detection of amplified DNA. These usually involve running the PCR amplified products on an agarose or polyacrylamide gel, containing a DNA intercalating dye and visualization of the gel in UV light.
The real time PCR differs from normal PCR as it does not require these post PCR procedures to be performed and the detection of amplified DNA is done by using a fluorescence reporter molecule, as the reaction progresses that is during each cycle. This fluorescence molecule emits fluorescence and this fluorescence increases as the reaction progresses. Thus, it allows real time monitoring of amplification. One of the other advantage of a real time PCR over normal PCR is that it can allow quantitation of DNA. Thus, it is also called quantitative PCR or qPCR.
There are two common procedures for detection of amplified DNA in real-time PCR. These are
1. Non-specific detection using dyes that non-specifically binds to dsDNA: In this approach, a DNA binding dye that specifically binds to dsDNA is used. The dye shows very little fluorescence when it is not bound. The fluorescence increases as dsDNA product accumulates during each cycle. Thus, DNA concentrations can be measured based on fluorescence intensity. One of the most commonly used dyes is SYBR green and it binds to minor grove of DNA double helix. Ethidium bromide can also be used. A major disadvantage of these non-specific dyes is that they bind to all ds DNA including primer dimmers.
2. Specific detection using sequence specific DNA probes: Another way of detection is using sequence specific oligonucleotide probes, which are labeled with a reporter dye at one end and a quencher dye at another end. Since, the reporter and quencher are placed very close, there is no fluorescence before the starting of reaction. During a PCR reaction, they are separated depending on their structures and thus the fluorescence is emitted. This fluorescence can be detected by the machine. The geometric increase in fluorescence corresponds to the exponential increase in product. This increase is used to determine the threshold cycle (Ct) in each reaction. Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. The quantity of DNA theoretically doubles every cycle during the exponential phase and relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles has 23 = 8 times more template after third cycle. Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA.
There are different probes available, including molecular beacon, TaqMan and Scorpion.
a. Molecular Beacon: It is single stranded and hairpin shaped. It consists of four parts. A single stranded loop that is complementary to the target sequence, a stem region which is made up of the two ends of the probe which are complementary to each other, a reporter dye at the 5’end and a quencher at the 3’ end. As the reporter and quencher are very close there is no fluorescence. When a beacon finds a suitable target sequence the stem portion of the beacon separates out and the loop hybridizes to the complementary target. As the two dyes become separated by this event, fluorescence is emitted.
b. Taqman Probe: A TaqMan probe is a specific oligonucleotide having quencher at one end and reporter at another end. During the PCR, the probe anneals specifically to an internal region (between the forward primer binding and reverse binding region) on the template strand. the 5' to 3' exonuclease activity of Taq polymerase breaks the proximity between reporter and quencher and the reporter molecule is released and thus fluorescence is emitted.
Real-time PCR consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software.
