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.