Multiplex PCR is a difficult technique that allows amplification of two or more products in parallel in a single reaction tube. This technology was first described by Chamberlain et al., in 1988, and has since been applied in diverse research areas, including analyses of deletions, SNPs, mutations, microsatellites, polymorphisms, pathogen identification, linkage analysis, gene expression, and forensic studies (Table).
Multiplex PCR, if sensitive enough, can be used to amplify and detect a single copy of a nucleic acid sequence and may also be used for both end-point and real-time PCR applications.
Multiplex PCR ensures standardization in certain experiments because identical reaction conditions and template amounts are used, pipetting and cycling condition variations are eliminated, and reliable comparison of results from a large number of fragments is achieved.
It also saves time and reagents for researchers performing large numbers of PCRs, hence its wide usage in various genotyping applications. However, certain challenges need to be addressed before the full potential of multiplex PCR can be explored.
Amplification of low copy-number target sequences in parallel with more abundant sequences is often limited by the generation of nonspecific PCR products and primer–dimers, and by the amount and quality of template DNA.
The establishment of optimal PCR parameters is the main factor influencing the success or failure of multiplex PCR. Traditionally, multiplex PCR has required extensive optimization of annealing conditions, enzyme amount, primer and probe concentrations, and buffer composition for maximum amplification efficiency of each target, leading to increases in costs and analysis time.
Compared with standard PCR systems using only two primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Competition for reagent resources and the resulting artifacts can be potential problems in multiplex PCR.
Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products. This often leads to unamplified DNA sequences and the absence of expected PCR products, eventually leading to false-positive or false-negative results.
In addition, traditional separation technologies (such as agarose slab gels) often do not provide sufficient resolution for post-multiplex PCR analysis and are not always optimal for separation of amplicons that differ by just a few base pairs.
Not addressing these bottlenecks leads to poor sensitivity, nonspecific amplification, and biased amplification of selected targets—challenges that scientists often encounter when initially setting up a multiplex PCR system, which involves several rounds of optimization with unpredictable success.