Advantages, Disadvantages and Application of Different Technics in PCR Technology

The basic analysis beside the conventional PCR reaction is a plus/minus assay, where the analyses indicates the presence or absence of PCR amplification of a fragment of a DNA molecule. Real-time PCR can monitor the amplification of the PCR product in “real time” with certain fluorogenic reporters. Two the most well-known reporters are SYBR green, which is actually a fluorescent dye that intercalarily binds to DNA, and the TaqMan probe system, which consists of an oligonucleotide probe that is complementary to a certain part of the DNA of interest. The aim of this study was to evaluate, compare and define the specifies of conventional PCR with SYBR green real-time PCR assays and TaqMan probe system. Real-time SYBR green has several disadvantages and advantages compared to the TaqMan probe system. The biggest advantage of the SYBR green is that any pair of primers can be used in this method, with the fact that the primers can be be adapted to real-time PCR technology. Also, the method is much more robust and financially profitable than the TaqMan assay system. The disadvantage of this method is linked to the advantage, because the method is less specific than TaqMan probes that are narrowly bound to a certain part of the DNA. Therefore, it is necessary to perform melting curve analysis after real-time PCR with SYBR green method. SYBR Green is relatively cost benefit and easy to use and technically based on binding the fluorescent dye to double stranded deoxyribonucleic acid (dsDNA) where TaqMan method has more expensive and based on dual labeled oligonucleotide and exonuclease activity of Taq polymerase enzyme. Specificity is the most important concern with the usage of any non-specific dsDNA-binding Dyes such as SYBR Green whiles more specificity showed by labeled oligonucleotide method such as TaqMan. The results of this study indicated that the developed conventional PCR and SYBR green real-time PCR assays are high sensitivity, specificity, accuracy and could be applied as an effective screening tool in different field of laboratory diagnostics.