How to Design PCR Primers for Multiplex Assays

Author: MJ Jasper

Good primer design is essential for successful, specific PCR amplification with high yield. For PCR primers to efficiently amplify DNA template in a PCR they should be carefully designed so the optimal conditions for them to amplify their target are as equivalently matched as possible.

There are many commercial and open source design software packages available to assist in optimal primer design, such as those provided on oligonucleotide manufacturing sites, including IDT and Eurofins Genomics, or Primer-Blast, Primer3, NetPrimer and PerlPrimer.  There is also extensive published information about primer design1-4.  Most of the primer features listed below can be achieved by defining the desired settings in primer design software.

An added layer of primer design complexity comes from amplifying more than one target sequence within the same PCR, or multiplexing.  During multiplexing, more than one target sequence is amplified by using multiple primer sets in a single PCR reaction.  This technique can have laboratory efficiencies resulting in time and reagent economies; however, it is particularly useful when sample template is limited to single cells or low-DNA template samples (eg biopsy testing using PG-Seq™ kits for PGT).  The more similar GC content, primer length, and primer melting temperature (Tm), the easier it is to scale up from a single primer set assay to a multiplex assay using the same PCR program.

PerkinElmer has developed a workflow combining whole genome amplification (WGA) and target amplification within the same PCR, called Target Sequence Enrichment or TSE.  This novel approach involves adding target specific primers for a single target or a multiplex primer pool for multiple targets part-way through the WGA program.  An aliquot of the resultant amplification product is then reamplified with either the same or a nested or hemi-nested primer set for the same targets.  Whilst the workflow sounds like it would require a highly complicated specific primer design, it does not.  The primers used in this workflow need to follow exactly the same specifications as any other primer.

The following features are recommended for designing primers in a multiplex assay.  If only a single primer set is needed for one target, then the parameters do not need to be quite as stringently adhered to.

Key primer features

Primer amplification specificity is partly dependent on the primer length and the melting temperature (Tm).

  1. PCR primers are generally designed to be 18 – 30 bp in length. Shorter primers anneal more readily to the target sequence, although they are also more likely to bind to more than one region in the genome and produce non-target amplicons.  For this reason, target-specific primers are usually longer.
  2. The melting temperature (Tm) of the primers should be between 58°C – 60°C, and all primers in the reaction should have a Tm within 0.5 – 1°C of each other.The base content impacts the Tm; G and C bases result in higher Tm’s than A and T bases. If challenges arise during primer design, consider the relationship of the primer sequence and length to Tm, and make suitable adjustments to ensure all primers are within the desired Tm range.
  3. The GC content of the primers should be between 40% and 60%. For template sequences with high GC content, primers with a higher Tm are recommended. Typically, Tm is calculated using the nearest neighbour calculation, however not all design software utilises this same calculation, so caution should be used when comparing primers designed using different packages.
  4. The 3’ sequence of the primer should not comprise more than 3 G or C bases in the last 5 bases. The 3’ sequence can end in a G or C base, as this promotes primer stability and binding. This is known as a GC Clamp.
  5. Avoid repeats of 4 or more of one base, or dinucleotide repeats (eg TGGGG or ATATATAT).
  6. Depending on the application and target sequence, the PCR generated amplicon length may range from 120 – 500 bp.

Ideally multiplex primers should generate amplicons of similar size and take into account the template length (whole cells, FFPE or fragmentated DNA template).  Consideration should also be given to the requirements of the downstream library preparation for Next Generation Sequencing (NGS). For example, if the primers are to be use with the PerkinElmer Target Sequence Enrichment (TSE) protocol and the amplicons will be sequenced using the recommended 1×75 bp NGS protocol, it will be important to consider whether fragmentation of the amplicon will be required during library preparation.  The need for fragmentation of the amplified target sequence will be dependent on the size of the amplicon and sequencing read length.  Another option is to extend the length of the sequencing reads.

Table 1: PCR primer design guidelines.

Primer Length18-30 bp18-30 bp
Melting temperature (Tm)56 - 62°C (within 2 - 4°C)58 - 60°C (within 0.5 - 1°C)
GC Content40%-60%40%-60% (optimal 50%)
Amplicon Length120-500 bp120-500 bp

Avoiding secondary structures

Typically, PCR primer design software will determine the degree of secondary structures and likelihood of primer dimer formation.  Additional resources such as MFEprimer, OligoEvaluator™ and the open source design software packages mentioned above may be useful if the primer design software does not evaluate secondary structures.

  1. Avoid hairpin structure formation.
  2. Avoid self-dimer and cross-dimer formation.

Modifications for other applications

Modifications to the 5’ sequence of primers are often made to accommodate the addition of an enzyme restriction site or linker sequence for PCR-indexing.  If these modifications are needed for the particular workflow being used, they will also need to be taken into consideration for the primer design.

Final primer design check

  1. Ensure each primer is specific to its target sequence and unlikely to generate additional (non-specific) PCR amplicons using Primer-BLAST, https://www.ncbi.nlm.nih.gov/tools/primer-blast/.
  2. Avoid primers annealing to known SNP’s, especially the 3’ sequence of the primer, using SNP databases and ensembl.org or Genome Brower and genome.ucsc.edu.

Evaluating primer performance

  1. For each individual target, a primer pair should generate a single amplicon of the expected size, with minimal primer-dimer. This can be confirmed using agarose gel electrophoresis, LabChip® microfluidic technology, or similar.
  2. Sequence the amplicon to confirm primer specificity.
  3. When individual primer pairs are multiplexed in a single PCR reaction, it is important to evaluate the breadth and depth of coverage of each primer pair for uniformity. Adjust individual primer pair concentrations in the multiplex pool if necessary, to balance the coverage of all primer pairs.


  1. Bustin S, Huggett J (2017) qPCR primer design revisited. Biomol Detect Quantif 14:19–28
  2. Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. PCR Methods Appl 3:S30–S37.
  3. Ozturk A, Can T (2017) A multiplex primer design algorithm for target amplification of continuous genomic regions. BMC Bioinform 18:306-314.
  4. Shen z, Qu W, Wang W, et al (2010) MPprimer: a program for reliable multiplex PCR primer design. BMC Bioinform 11:143-149.

Recent Posts



Recent Tweets

For research use only. Not for use in diagnostic procedures.