Multiplex PCR

What is Multiplex PCR?

Multiplex polymerase chain reaction (PCR), first reported in 1988, is a type of PCR in which two or more target sequences can be amplified simultaneously by including more than one pair of primers in the same reaction. This technique requires two or more probes that can be distinguished from each other and detected simultaneously. There are different probe technologies available, all of them use fluorophores. Multiplex PCR has been established as a general technique, applied in SNP genotyping, pathogen detection, GMO (genetically modified organism) detection, forensic studies, food analysis, mutation and polymorphism analysis, gene deletion analysis, template quantitation, etc. Compared with conventional PCR, multiplex PCR has many advantages, including more information with less sample, higher throughput, cost effective (fewer dNTPs, enzymes, and other consumables), time saving, less input material required, more data from limited starting materials, increased accuracy of data normalization and fewer pipetting errors. Although the technique is advantageous, the multiplexing is not applicable to all types of reactions. For the larger amplicon such as 800bp or 1000bp, multiplex PCR might not work efficiently always.

General Process of Multiplex PCR

Multiplex PCR is composed of multiple primer sets in a single PCR mix to generate amplicons of different sizes specific to different DNA sequences. By targeting multiple sequences at once, additional information can be obtained from one test run. The annealing temperature of each primer set must be optimized to work properly in a single reaction, and when viewed by gel electrophoresis, the amplicon size should have a large difference to form different bands. In addition, if the amplicon sizes overlap, primers that have been stained with different colors of fluorescent dyes can be used to distinguish and visualize different amplicons. Multiplex PCR can be divided into two categories: multiple template multiplex PCR and single template multiplex PCR.

Multiple Template Multiplex PCR

Multiple template multiplex PCR uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates. It can be used in the detection of different strains or species of pathogens. However, this technique is not valid for the detection of inherited genetic disorders.

Mechanism of multiplex multiple  template multiplex PCR Fig. 1 Mechanism of multiplex multiple template multiplex PCR

Single Template Multiplex PCR

Single template multiplex PCR uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template. Different from multi-template multiplex PCR, single template multiplex PCR can be used in the detection of inherited genetic disorders. It’s also widely used in the deletion analysis and genotyping.

How to Perform Multiplex PCR

Developing a multiplex PCR assay is not easy because it requires high expertise and trial and error experiment runs for one particular multiplex reaction. Developing some multiplex PCR systems may be as simple as combining two sets of primers for which reaction conditions have been confirmed. However, other multiplex PCRs must be developed considering the regions to be amplified, the relative sizes of the fragments, the dynamics of the primers, and the optimization of PCR technique to accommodate multiple fragments. The step by step process is as following:

  1. Choose Loci, determine the PCR system, distribute amplicons (localized at mutation hot spots, linked to genes, chromosomally unlinked, grouped close exons in a single amplicon, etc.). Internal control fragments (other exons, external sequences, host sequence, sequence conserved in all target templates, etc.) should be designed. The regions selected for multiplex amplification can be determined by the nature of the analysis; for example, deletion assays amplify exons, forensic assays distinguish individual variation at highly polymorphic markers, and microbial assays may exploit strain- or species-specific variation.
  2. Position primers in regions of detailed sequence (It’s related to amplicon sizes). Detailed sequence information for primer sites at the selected loci is important, because nonspecific amplification may occur at other sites with similar sequences, or reduced amplification may occur at primer-template mismatched sites.
  3. Design primers with similar reaction kinetics.
  4. Develop PCR conditions separately for each primer set. Thermocycling parameters are also determined mainly by the sequence of the primer sets.
  5. Add primer sets sequentially, altering conditions as necessary. Reduce nonspecific amplification (hot start, ionic detergents, short extension times, hottest annealing, reselect primer sequence). Vary relative concentrations of primer sets for equal amplification. Change buffer systems if necessary.
  6. Adjust reaction components and cycling conditions for multiplex amplification. Mg2+, dNTP, and polymerase requirements may increase. Ideal extension times may be longer.

After these steps, Analysis like gel electrophoresis of multiplex products is requisite for some systems. Many of the techniques for product analysis developed for uniplex PCR can be applied directly to multiplex PCR.


  1. Edwards M C, Gibbs R A. Multiplex PCR: advantages, development, and applications[J]. Pcr Methods & Applications, 1994, 3(4):65-75.
  2. Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: A practical approach[J]. Journal of Clinical Laboratory Analysis, 2002, 16(1):47-51.
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