Probes and Dyes used in Real time PCR assay; a) SYBR Green; b) TaqMan; c) Molecular Beacon; d) Scorpions 

Probes and Dyes used in Real time PCR assay; a) SYBR Green; b) TaqMan; c) Molecular Beacon; d) Scorpions 

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Quantitative Real-time Polymerase Chain Reaction (qPCR) is an important tool for molecular biology and biotechnology research, widely used to determine the expression levels of mRNA. Two main methods to performing qPCR are largely used: The absolute quantification, in which the mRNA levels are determined by using a standard curve and the relative m...

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Context 1
... dye is used to monitor the amplification of any DNA sequences and dispenses the use of a probe, thus reducing the cost of amplification and providing a great advantage in its application. On the other hand, since the dye binds not only to the target DNA, but to all dsDNA formed during qPCR, the use of SYBR Green, while simple lacks specificity (Figure 1a). The specificity of the reactions, however, can be easily accessed by the use of melting curve analysis ( Dheda et al., 2004). ...
Context 2
... probe is complexed with a quencher and a reporter fluorophore dye at its 3' and 5' ends, respectively (Livak et al., 1995). During the amplification step the probe is associated to its complementary target DNA and then is cleaved by Taq DNA polymerase 5'-3' exonuclease activity (Figure 1b). This cleavage releases the reporter dye and generates a fluorescent signal that increases with each cycle ( Bustin et al., 2002). ...
Context 3
... reporter fluorescent dye and the quencher remain extremely close and therefore no fluorescence is detected when www.ccsenet.org/jmbr Journal of Molecular Biology Research Vol. 5, No. 1; this structure is formed (Figure 1c). However, during the annealing step, Molecular Beacon hybridizes to the target sequence generating conformational changes leading to the separation of reporter and quencher dyes, which results in the emission of fluorescence ( Tyagi et al., 1996;VanGuilder et al., 2008). ...
Context 4
... consist of a single-stranded oligonucleotide probe of approximately 20 to 25 nt carrying a reporter fluorophore at its 5' end and a quencher at its 3' end. Their tridimensional conformation resembles a stem and loop structure, in which a PCR primer is attached ( figure 1d). The stem-and-loop structure acts as a blocker to prevent DNA polymerase activity during the interaction of the probe with the target DNA ( Bustin et al., 2002;Ng et al., 2005). ...

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