Multiple targets, one run: multiplex your PCR!

What the heck was multiplex PCR?

Although we have heard the term a thousand times, there are often uncertainties what is behind it. Maybe you already do multiplex PCR without naming it that way? Here you go: A multiplex PCR amplifies multiple targets within a single PCR run, in a single reaction vessel, from a single sample aliquot - instead of performing many separate reactions. Or, in other words, targeted amplification of multiple genes. You use the same reagent mix, but each target region has its own primer set. Multiplex PCR is used for genotyping, forensics, food testing, pathogen detection, polymorphism analysis and many more applications. It is also part of many targeted sequencing approaches in next generation sequencing (NGS) protocols.

New benefits with an “old” method

If you stick to singleplexing but wish to get a variety of information from the same sample, you have to perform multiple runs. Multiple runs mean more sample material, more reagents, more time. Accuracy, precision and comparability can suffer, as assays are performed in different vessels with different reaction conditions and with a higher risk of pipetting errors.

One run with various amplicons instead of several runs - the advantages of multiplexing are undoubtedly obvious:

• Increased productivity

Saves precious time for repeated experiment setup/analysis and enhances throughput.

• Higher information value

More data from just one sample.

• Saves costs and sample material

Sample material is required only once to amplify all desired target regions. Likewise, PCR-reagents and consumables, which saves your budget.

• Increased accuracy and comparability

Multiplex PCR makes reaction conditions absolutely comparable, because they are identical for each amplicon. Slight differences are not significant as genes to be compared are amplified in the same well.

• Improved precision

Pipetting once instead of several times minimizes pipetting errors.

• No investment in a new cycler and consumables

…if you have the right ones!

Putting the cards on the table

Establishing and optimizing the new assay is more complex than a singleplex PCR. Preventing unspecific priming, underrepresented products and loss of yield are key in assay set-up. Typical measures include: primer design with in silico tools, the avoidance of dNTP-depletion ensured by equally abundant templates, optimizing and matching the individual primer combinations with regard to the PCR conditions. Did you know, that thermocycler and consumables are equally important influencing factors?

The impact of thermocycler and consumables

Cycler and consumables can lead to non-specific PCR products, loss of yield and can affect the reproducibility of results. Temperature differences and too high or too low temperatures after heating or cooling ramps (over- and undershoot) can reduce PCR yield and increase the risk of mispriming. Plasticizers, biocides or slip agents in plastic-materials can falsify results.

You can avoid that:

1. Guarantee temperature stability & a low over- and undershoot

Precise ramp rates and stable temperatures avoid unspecific amplifications and allow maximum yields. Likewise, the cycler should control that temperatures are within optimal range, i.e. not too high or too low. Temperature stability and precisely controlled temperatures increase the polymerase’s half-life. The enzyme will thank you with a better yield and drastically reduced misprimed secondary products.

2. Go for leachable-free & clean consumables

Ensure to use “clean” PCR-plates, which are free from leachables, nucleases, DNA or PCR-inhibitors. By the way, thin vessel walls guarantee a perfect temperature transfer. To facilitate that more molecules are available for amplification, use plates where fewer molecules adhere (low binding plates).Knowing pitfalls and the way out, establishing a multiplex PCR protocol is not as complex as you might think!

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