Quantitative real-time PCR (qPCR) is a sensitive and robust technique directly evolved from the “end-point detection” polymerase chain reaction (PCR). PCR is a polymerase-dependent repetitive thermal reaction able to generate copies of a specific template spanning from two short oligo-deoxynucleotide sequences (primers). The qPCR is at least 100-fold more sensitive than the end-point detection and displays a dynamic range not less than nine logs.

In general, PCR is based on the quantitative relationship between the amount of target sequence at the beginning and the amount of amplifi ed PCR product at any given cycle. Such correlation follows an exponential rate that gives rise to an exact doubling of product that is accumulated at every cycle (when 100 % reaction effi ciency is assumed). Such exponential phase is limited to a short number of PCR cycles since the reaction occurs in a classical closed system. This state causes the depletion of reactants concentrations, enzyme activity, and other factors, while the products accumulate over time. Thus, the PCR is characterized by four reaction phases known as: Baseline, Exponential, Linear, and Plateau. Baseline is very short step where the amplifi cation is not yet detectable. During the second phase of amplifi cation the kinetic of reaction determines a favorable doubling of amplicons. Linear phase is characterized by slowdown trend of amplifi cation and the products are no longer doubled at each cycle. Finally, at the plateau the reaction is essentially terminated , no more accumulation of amplicons is achieved even if the number of cycles is increased and, very unuseful, PCR products may start to degrade. Traditional PCR detects reaction products at such last phase, thus it is so called “end-point PCR.” Worthily, each reaction can reach the plateau at a different point and may be characterized by different kinetics that leads to different performances. Thus, end-point PCR cannot be used for quantifi cation purposes. Contrary to the end-point PCR, the qPCR allows the immediate detection of amplifi ed products at any given cycle using a quantitative relationship with the target sequence at the beginning of reaction.

Real-time detection should be performed during the exponential phase where the fl uorescence signal is directly proportional to DNA concentration.

Two essentially different types of chemical strategies ensure such generation of fl uorescent signal. One is based on doublestranded intercalating dye (SYBR-Green and its evolution) and the other can use a plethora of different dye-labeled probe systems (i.e., exonuclease-based double-labeled dye oligo- deoxynucleotide, molecular beacons). In general, qPCR detection achieved using intercalating dye is defi ned as “nonspecifi c,” while detection by fl uorescent probes is considered “template-specifi c”. Such dichotomy assumes that the use of probe introduces an additional level of specifi city since it does not produce any fluorescence signal, due to probe hybridization, for amplicons generated by either mis-priming or primer-dimers.

On the implementation of qPCR point of view a new powerful technique called HRM (high-resolution melting) has also been developed. Such technique is a postamplifi cation analysis that uses data acquired at the plateau phase in order to accurately quantify mutations, polymorphisms, and epigenetic differences on doublestranded nucleic acid molecules. It is based on the use of both saturating dye and instruments that are able to monitor tiny differences of melting temperature of the double strand. HRM is an excellent alternative to the classic molecular methods for screening genetic variants such as dHPLC sequencing.

Thus, it confi rms continuous improvements of qPCR along its two decades of life leading to novel technical evolutions.

While the number of its applications is increasing exponentially as the mechanism of PCR itself, there is neither consensus on experiment design nor homogeneity in practice. Therefore, in order to achieve reliable experiments and unequivocal interpretation of qPCR data, several practical guidelines have been recently proposed.

The third generation of the PCR is the digital PCR (dPCR) that should even be considered a modifi ed qPCR showing high sensitivity that allows an absolute quantitation. In fact, it is a hybrid application using a classic PCR reaction together with fluorescencebased detection. Both the extreme dilution and partitioning of the sample yield to produce single-molecule subreactions, some of which carry target sequence while others do not; such ratio is used to quantify the starting amount of the target template.

Therefore, the wide range of possible applications of qPCR, such as clinical diagnosis, molecular research, and forensic studies, already make it a mature but not outdated technology.