The polymerase chain reaction (PCR) is a well-established amplification technique that is widely used to rapidly make millions of copies of a specific DNA sample. It was developed by Kary Mullis in the 1980’s and is based on the ability of DNA polymerase to synthesize a new strand of complementary DNA from a template strand. A primer is needed to add the first nucleotide, as the polymerase enzyme can only start making the new complementary DNA strand once the primer has bound. Therefore, primers act as the starting point for DNA synthesis.
For more information about the advent of PCR methods that have enabled the detection of nucleic acids in small sample sizes, check out the Next-Generation PCR ONLINE webinar series on-demand.
Multiplexing is a term that refers to multiple samples being processed at the same time, usually to save time and money. Multiplex PCR is a technique whereby PCR is used to amplify several different DNA sequences simultaneously. It is a type of target enrichment approach. It was first described in 1988 as a method to detect deletion mutations in the dystrophin gene – the largest known human gene. The dystrophin complex connects the cytoskeleton of muscle cells with the extracellular matrix, which consists of proteins and other molecules outside of the cell. In 2020, real-time-PCR multiplex assays were designed as a method for increasing the SARS-CoV-2 diagnosis capabilities by combining multiple gene targets into a single reaction.
What are ChIP-seq and bisulfite sequencing?
How does multiplexing work?
Multiplex PCR consists of numerous primer sets within a single PCR mixture. This means that the products produced are of varying sizes and are each specific to different DNA sequences. Multiplexing reactions can be divided into two categories – single template and multiple template reactions. Both still use several primers.
Diagram showing the multiplex PCR approach, whereby all of the primers are added to a single tube. Image credit: S. Rosel, 2021
Annealing temperatures for each of the primers must be optimised to work in a single reaction. Therefore, the design of specific primer sets is essential for the success of the multiplex reaction. Primers are usually about 18-22 base pairs long and have a melting temperature of 55-60°C, unless the sequences have high GC content. In this case, the melting temperature is higher. Specificity and dimerization should also be considered when designing the primers. Another requirement is enough variation of base pair length so that distinct bands are formed during gel electrophoresis.
The advantages of multiplex PCR
The benefits of multiplex PCR can be substantial in labs where repeated analysis of similar types of samples and the same targets is required. Advantages include gaining more information with limited starting materials, its cost-effective nature, time saving and higher throughput. Essentially, by targeting multiple sequences at once, additional information can be gained from a single test run that may otherwise have needed a additional reagents and more time to perform.
Also, false negative results are often revealed in multiplex assays, which may not have been detected in simple PCR. This is because each product provides an internal control for the other amplified fragments.
There are a number of companies that sell kits for multiplex PCR, including Qiagen and Agilent. These kits have been useful in a number of applications, including DNA genotyping, whole genome amplification analysis and cell line identification. They are ready-to-use – all the necessary reaction components and appropriate primer sets are provided. Qiagen’s multiplex PCR kit works with up to 16 primer pairs and is useful for typing transgenic organisms or microsatellite analysis. Agilent has optimised an approach called hybrid capture-based target enrichment, which amplifies more than 100 fragments simultaneously.
More information about these (and other) multiplex PCR kits can be found in our Sample Preparation report. It includes specifics about various kits that are developed by a number of companies:
The disadvantages of multiplex PCR
Although multiplex PCR has many advantages, it also has several disadvantages that cannot be ignored. Firstly, self-inhibition among different sets of primers can occur. Primer dimers are a potential by-product of PCR, which consist of two primer molecules that have hybridized to each other because they share a string of complementary bases. DNA polymerase then amplifies the primer dimer, competing for the PCR reagents, and potentially inhibits amplification of the target DNA sequence. Therefore, primer dimers cause the second disadvantage, which is low amplification efficiency. Generally, this makes multiplex PCR challenging to design and means that the technique cannot be scaled easily to very large targets.
Splitting up samples is often required to reduce the challenges faced by large sample sizes. However, this in turn decreases the tests sensitivity to rare variants. Using droplets or linking primers to increase specificity are alternative solutions that have been used to overcome some of these obstacles and reduce primer dimer formation.
Another complication is that only a single set of amplification conditions can be set for each PCR product. Therefore, primer design for some multiplexed targets can be difficult. There is a risk of favouring certain products over others, creating biases. An option is to consider several factors during primer design, such as target nucleic acid sequence, length and GC content. This makes it more likely that the set of amplification conditions will be optimal for all of the products that are targeted by the multiplex PCR. Most researchers now use in silico design tools, on computers, to simplify these tasks.
An Overview of Targeted Sequencing
Image credit: BioSpectrum Asia