The components needed for a basic PCR reaction are: DNA/RNA template containing a sequence that needs to be amplified Two oligonucleotide primers (small DNA fragments) dNTP (deoxynucleotide triphosphates) Heat stable polymerase (enzymes) Magnesium ions in buffer The PCR reaction is performed with repeated fluctuations of the temperature in each cycle. There are three main temperature steps in each cycle, such as denaturation, annealing, and elongation. In this step of the PCR reaction, a high temperature of 95-98 is applied to melt During annealing the temperature is lowered to 50-65 to let primers anneal to each of the single-stranded DNA sequences. In the third step temperature of 72 is usually used. In this phase, a new DNA strand is synthesized which is identical to the DNA template strand.
Real-time PCR is a technique for quantitative nucleic acids analysis, which is simultaneously amplifying DNA sequences and detecting if a certain DNA sequence (e.g. virus or bacteria) is present. This technique results with copying (amplifying) one sequence to many thousands of copies. The replication of DNA is in real time, hence RT-PCR allows to accurately measure the amount of product accumulated in each cycle of the reaction. The increase of the fluorescence signal is used as an indication that the number of replicated sequences has also increased .
Furthermore, the real-time monitoring allows collecting data as the process goes on.The amplification curve is generated by plotting the fluorescence level in each cycle of the process, where the amplification efficiency can be estimated. One real-time PCR reaction is defined with around 40 cycles, and in each cycle, all three steps (denaturation, annealing, elongation), previously mentioned, are performed. One amplification curve as shown in figure , can be separated into three characteristic regions, representing one real-time PCR reaction. Baseline – or the initiation phase is defined as the fluorescence level measured before detecting any amplification. Usually during this phase, the fluorescence values are low, resulting in a horizontal line close to the x-axis. During this initial cycles, usually 3-15, the background noise is much stronger than the fluorescence signal and is causing interference, resulting with a not reliable recording of the fluorescence signal.
Exponential – Early in the exponential phase the fluorescence signal is reaching a threshold, usually called where the signal of the fluorescence is higher than the background noise . After this threshold, the reaction efficiency is exponentially increasing and the initial quantity of the sequence that is being amplified can be measured, based on the amount of fluorescence in each cycle. Plateau – In this phase the reaction efficiency is dropping, and the signal is leveling off. The reason for this might be that the reaction is running out of components, such as reagents, or it might be that there is a conflict between two processes in the reaction Another reason can be the conflict between primer and amplicon as stated by