Theoretical basis of multiplex PCR method: Critical Parameters
Multiplex polymerase chain reaction (PCR) is a generation of PCR in which two or more genes are amplified in the same reaction simultaneously. Since 1988, when it was first described (Chamberlain et al., 1988), This method has been effectively employed in various fields of DNA testing, including deletion analyses (Henegariu et al., 1994), mutation analyses (Shuber et al., 1993), polymorphism analyses (Mutirangura et al., 1993), quantitative assays (Mansfield et al., 1993), and reverse transcription PCR (Crisan, 1994). The role of many parameters that may influence standard (uniplex) PCR performance has been discussed (Robertson & J., 1998). However, there are fewer papers that discuss multiplex PCR (Henegariu et al., 1997). Multiplex PCR optimization might be problematic due to a lack of sensitivity or specificity, as well as preferred amplification of specific targets (Polz & C. M., 1998). The presence of more than one primer pair in a multiplex PCR increases the likelihood of receiving erroneous amplification results, owing to primer dimer formation (Brownie et al., 1997). These nonspecific products may be amplified more effectively than the desired target, consuming reaction components and causing annealing and extension rates to be slowed. As a result, the goal of multiplex PCR optimization should be to minimize or eliminate non-specific interactions. It is critical to ensure that the primers in the reaction mixture are compatible so that no interference occurs. Simple rules were used to select primers: I primer length of 18–24 bp or greater; (ii) GC content of 35–60%; and (iii) annealing temperature of 55–58 °C or greater. Longer primers (28-30 bp) allowed for a higher annealing temperature, which resulted in fewer unspecific products. Changing/optimizing several of the reaction parameters was required to combine the primers in various mixes and amplify many loci at the same time. When performing a multiplex reaction for the first time, it’s best to use equimolar amounts of primers. The findings will indicate how specific primer concentrations and other parameters should be adjusted. Special attention to primer design parameters such as homology of primers with their target nucleic acid sequences, their length, the GC content, and their concentration have to be considered (Robertson & J., 1998). (Robertson & J., 1998). In a multiplex PCR, all primer pairs should ideally permit equivalent amplification efficiency for their respective targets. This can be accomplished by using primers with approximately equal optimum annealing temperatures, and they should not be homogeneous internally or to one another (Henegariu et al., 1997). The extension rate of specific primer-target hybrids is also affected by the enzyme’s activity, the availability of critical components such deoxyribonucleoside triphosphates (dNTPs), and the target DNA’s nature. As a result, the majority of modifications to increase PCR performance have focused on parameters that affect annealing and/or extension rates. As more loci are simultaneously amplified in multiplex PCR, the pool of enzyme concentrations, PCR buffer ingredients, and nucleotides becomes a limiting factor, and the polymerase molecules require more time to finish synthesis of all the products. Variation in reaction component concentrations above those utilized in uniplex PCR is most probable due to the competitive nature of the PCR process. The more efficient amplification of alternative targets (including nonspecific products) can outcompete the desired target DNA, lowering the efficiency of the desired targets’ amplification and thus the reaction’s sensitivity (Raeymaekers, 1995).
When used in quantities ranging from 5% to 10% (vol/vol), some researchers suggested dimethyl sulfoxide (DMSO) plus glycerol to improve PCR amplification efficiency (more product) and specificity (no unspecific products). In multiplex PCRs, bovine serum albumin, or betaine, has also been reported to be beneficial (Jackson et al., 1996). The components may prevent DNA polymerization stalling, which can occur during the extension process when secondary structures form within areas of template DNA (Hengen, 1997). It can also serve as destabilizers, decreasing the melting temperature of GC-rich sequences, or as osmoprotectants, boosting the polymerase’s tolerance to denaturation (Hengen, 1997). The use of hot start PCR (Chou et al., 1992) and/or nested PCR has proven a simple answer to issues faced in the development of multiplex PCR. Before commencing thermocycling, the former frequently eliminates nonspecific reactions (notably the generation of primer dimers) induced by primer annealing at low temperatures (4 to 25°C) (Chou et al., 1992). The procedure has recently been made more practical by the use of a non-mechanical hot start methodology that involves the use of a form of Taq polymerase, such as Ampli Taq Gold (Roche Diagnostics), that is activated only if the reaction mixture is heated to approximately 94°C for 10 minutes in the first denaturation step (Kebelmann-Betzing et al., 1998). Through two distinct rounds of amplification employing two discrete primer sets, nested PCR improves the test’s sensitivity and specificity. Although this adaption is unquestionably beneficial in the vast majority of cases, it significantly complicates PCR’s practical deployment. The second stage of amplification slows down and amplifies the data.