Loading
Home | News | Multiplex PCR: Practical guidance for primer design and assay optimisation

Multiplex PCR: Practical guidance for primer design and assay optimisation

Regardless of whether you’ve done a multiplex PCR before, or are just about to start, we’ve put together this short guide with practical advice on setting up such assays.

1. What is multiplex PCR?

Multiplex polymerase chain reaction (multiplex PCR) is a modification of conventional PCR that uses multiple primer pairs to amplify two or more target DNA sequences simultaneously in a single reaction tube. Since its introduction in the late 1980s, multiplex PCR has become a valuable technique for rapidly detecting multiple genetic targets while reducing reagent consumption, sample usage and analysis time¹,².

2. Advantages of multiplex PCR

  • Simultaneous amplification of multiple targets in a single reaction.
  • Reduced reagent consumption.
  • Lower template DNA requirements.
  • Increased laboratory throughput.
  • Fewer pipetting steps and handling errors.
  • Inclusion of internal amplification controls within the same reaction.
  • Lower overall assay costs resolved.

3. Limitations of multiplex PCR

  • Competition between primer pairs.
  • Competition between amplicons for polymerase activity.
  • Unequal amplification efficiencies.
  • Increased likelihood of primer-dimer formation.
  • Greater sensitivity to suboptimal primer design.
  • More demanding optimisation requirements.

Multiplex PCR therefore requires further optimisation, rather than simply combining multiple singleplex reactions in one tube.

Figure 1. Schematic of a multiplex PCR reaction. In this example three different targets are amplified from the same sample in a single reaction tube. 1. Primers are designed to specifically amplify three different targets with sufficient separation (on agarose gels) or combined with target-specific, spectrally distinct qPCR probes (for qPCR). 2. One PCR reaction mix is set up with one sample and combining all three primer pairs. 3. Optimised cycling conditions enable the amplification of all three targets, which can be resolved on an agarose gel.

4. Primer design for multiplex PCR

In addition to standard primer design requirements, all primer pairs should ideally meet the following criteria:

  1. The melting temperatures (Tm) of all primer pairs should generally be within approximately 2–3°C of one another. Large differences in Tm make it difficult to select a single annealing temperature that supports efficient amplification of every target.
  2. Primer pairs should have comparable amplification efficiencies. Highly efficient primer pairs can rapidly consume reagents, suppressing amplification of weaker targets.
  3. Minimise primer interactions. Analyse every primer against both its intended partner and every other primer in the reaction. Because the number of possible primer interactions increases rapidly with each additional primer pair, computational analysis is essential during assay design.
  4. Amplicon size separation. For endpoint PCR analysed by agarose gel electrophoresis, products should differ sufficiently in size to allow clear separation. As a general rule, minimum recommended separation for 1% agarose is ~80–100 bp and for 2% agarose, ~30–50 bp. See the table below for an example.
Target Example length
Target 1 180 bp
Target 2 300 bp
Target 3 450 bp
Target 4 650 bp

5. Eight practical recommendations for multiplex PCR3

  1. Design each primer pair according to standard PCR principles in combination with the extra tips listed in Section 4 above.
  2. Validate each primer pair individually in singleplex reactions.
  3. Confirm comparable amplification efficiencies.
  4. Screen for primer-primer interactions using primer design software.
  5. Combine primer pairs incrementally rather than all at once.
  6. Optimise primer concentrations to balance product yields.
  7. Optimise magnesium concentration and annealing temperature.
  8. Validate the final assay using representative positive and negative samples.

6. References

1. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., & Caskey, C. T. (1988). Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research, 16(23), 11141–11156. https://doi.org/10.1093/nar/16.23.11141

2. Edwards, M. C., & Gibbs, R. A. (1994). Multiplex PCR: Advantages, development, and applications. Genome Research, 3(4), S65–S75.

3. Markoulatos, P., Siafakas, N., & Moncany, M. (2002). Multiplex polymerase chain reaction: A practical approach. Journal of Clinical Laboratory Analysis, 16(1), 47–51.

 

 

Other News