“Multiplex PCR amplifies multiple DNA templates regions, simultaneous using a different set of primers in a single PCR reaction.”

PCR technologies are utilized so often in diagnostic and research organizations. It is efficient and accurate but experimental cost, time-consuming and tedious processing of it, is a major set back, though.  It takes approximately 3 to 4 hours from DNA to results interpretation for a single reaction.

Minimizing the cost and time duration for PCR is quite difficult for us but what if we perform multiple experiments in a single reaction? It saves money and time. 

And that exactly multiplex PCR facilitates.

Various template region amplifies in a single PCR run, means, in 4 hours we can perform multiplex PCR assays using multiplex PCR technique. It isn’t a different machine, it is just a modification in conventional PCR.

The technique was first described in the year 1988 by Jeffrey S. Chamberlain and coworkers. Various dystrophin gene locus of the muscular dystrophy patient had been amplified by them. Multiple deletions of a dystrophin gene had been detected by utilizing various sets of primers. 

Scientists use mPCR (multiplex PCR) in virology, microbial genetics, and single gene disorder studies. Also, It has the potential for prenatal screening of various genetic disorders.

In the present article, we will understand the whole concept of the multiplex PCR and also learn the process of how to do it.

Major topics covered in the article are,

  • What is multiplex PCR
  • How to develop a multiplex PCR assay
  • Our protocol of multiplex PCR
  • Advantages of multiple PCR
  • Disadvantages of multiplex PCR
  • Application of multiplex PCR

What is a multiplex PCR?

“More than two sets of primers amplify several different sequences of the templet DNA or sequences of multiple temples in a single PCR reaction is known as multiplex PCR.”

In a simple language, we can say, it is a combination of different PCR reaction thus more reagents are needed to amplify various template regions. Here also, general PCR reagents like dNTPs, reaction buffer, nuclease-free water, and Taq DNA polymerase are used.

The benefit of doing multiplexing is to save time. However, more amount of each reagent required to succeed in the reaction.

Based on the template DNA the multiplex PCR can be divided into two categories:

  1. Multi-template multiplex PCR
  2. Uni- template multiplex PCR

The multi-template multiplex PCR is slightly different than the uni-template. Here, different templates are simultaneously amplified in a single PCR reaction. Different sets of primers are used to do so. However, it does not mean to pool different samples in a single reaction.

The present technique facilitates the detection of various pathogens from a biological sample. Also, using sequence-specific primers of the same pathogen various strains of the pathogen can also be determined.

Therefore, the multi-template multiplex PCR is not a valid technique for the detection of inherited genetic disorders. 

What is a multiplex PCR?

The image represents the multi-template multiplex PCR

On the Otherside, the uni-template multiplex PCR is used in the detection of inherited genetic disorders. Here our aim is to amplify various genes or regions from a single sample. For instance, if we wish to detect all five mutations of thalassemia, our template is a single beta-globin gene from a single patient. But we are analyzing 5 different alterations in a beta-globin gene. 

The single template is amplified with the help of the multiple sets of primers in a single reaction. The single template multiplex PCR is widely used in the deletion analysis and genotyping.

What is a multiplex PCR?

The image represents uni-template multiplex PCR

Read more on PCR,

  1. What is immuno-PCR or IPCR?
  2. What is ARMS-PCR or allele-specific PCR?
  3. A Complete Guide of the Polymerase Chain Reaction

Although it is a simple set of PCR, two factors are very crucial in designing the multiplex PCR.

  1. Primer designing
  2. Reagents used in the PCR reaction

The length of primers, specificity, melting temperature, and primer-dimer formation capabilities are important considerations of primer designing. Read our article: PCR primer design guidelines.

The quality and quantity of reagents also make a difference. If we use a higher amount of reagents, it amplifies non-specific regions as well. Use a desirable amount of each reagent (the quantity of reagents differ in the different assay).

How to develop a multiplex PCR assay?

Unlike conventional PCR, mPCR or multiplex PCR need higher expertise and experience to validate each assay. Furthermore, vast research, trial and error experiments, and standardization runs needed to develop various multiplex kits.

Need some extra starting material, unlike the normal PCR. for example, a highly concentrated or higher amount of template DNA needed during single template multiplex PCR.

Likewise, more units of Taq DNA polymerase and PCR reaction buffer is required, again, an inappropriate quantity of reagents fail our experiment.

Here, primer designing is a crucial and very important process.

Because we are using more than two sets of primers, all the primers must be different from one another. The primers should be non-complementary to one another to minimize primer-dimer formation. This means, that each set of primers should be unique and different. 

With this, the GC content and melting temperature should be between 45% to 60% and 55°C to 60°C, respectively. Following these criteria increases assay success chances. 

Let me give you some technical information regarding multiplex primers. To achieve higher amplification, the annealing temperature of each set of primers should be nearer to one another, ideally. Further, the length of each primer should not be more than 30 nucleotides, ideally, 20 to 25 nucleotide long primer is recommended.

Yet another crucial factor is the PCR cycles. More PCR cycles can also cause reaction failure or truncated amplification as fewer reagents are available in later cycles. Contrary, if less PCR cycles are set, amplification aborted prematurely, we can say, not all the regions amplify properly.

Conclusively, use proper cycling conditions, that is why I had told you that designing multiplex PCR needs high expertise and experience.

25 to 30 cycles are sufficient for it.

Now, our multiplex PCR assay is ready to perform.

Protocol for multiplex PCR:

The multiplex PCR is a very sensitive reaction, every experiment needs different setup and reagent requirements.

However, some simple templates can be amplified using the same reaction setup of simple PCR, for example, to perform the Y chromosome microdeletion multiplex of 6 different microdeletion studies, we need a single conventional PCR set up and reaction preparation.

Contrary, for some tougher templates, like the DMD, more complex PCR set up and a high amount of reagents and reaction enhancers are needed.

The ideal concentration of each component used in the multiplex PCR are,

Component Concentration Quantity
Master mix 1X 13µL
PCR reaction buffer 1X If needed
Forward primer (four sets of primers) 10pM 1 x 4= 4µL
Reverse primer (four sets of primers) 10pM 1 x 4= 4µL
Template DNA 50ng 5µL
Water 4µL
Total ——————————- 30µL

(Note: the ready to use mastermix contains the PCR buffer, so the PCR reaction buffer is not needed).

The reaction conditions are,

PCR Steps Initial Denaturation Denaturation Annealing Extension Final extension
Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-6o ̊C 72 ̊C 72 ̊C
Time 5min 1min 50sec 1min 7 min
——————– ——————- 25-28 cycles ————— ———————

To enhance the amplification capacity and accuracy, we need various other reagents like KCl, MgCl2, DMSO or albumin. Non-specific bindings, primer-dimers, hairpin formation, and other PCR problems can also be encountered using PCR enhancers. 

Read more,

DNA sequencing Gene editing

Advantages of multiplex PCR:

  • The present method is accurate and rapid, amplify different templates in same time.
  • Also, less manpower is needed, thus the present method is cost-effective.
  • Each amplicon works as an ‘internal control’ for other amplicons, therefore, the chance of false-positive results is less.
  • By comparing different amplicons of a single template we can determine the quality of the template.
  • We can get more information by using low samples.
  • In terms of technical advantages, here the pipetting errors are less and less consumables, and chemicals required to perform an experiment.

 Limitations of multiplex PCR:

  • longer multiple templates can’t be amplified using the present method. Usually, 1000bp or more gene regions can’t properly be amplified.
  • It is restricted to a few types of template, we can’t do multiplex for all types of reactions. 
  • Further to this, the chance of reaction failure and non-significant results are commonly observed in multiplex PCR.

Application of multiplex PCR:

The multiplex PCR is broadly used in the virology and pathogen detection.

In modern-day science, the present method is widely used in microbial detection and identification, and virology studies. Traditional microbiology culture techniques are tedious, time-consuming, and prone to contamination.

Multiplexing revolutionized the PCR technology due to its accuracy, precision and rapid nature. various pathogens or strains of pathogens and viruses can be identified from different biological samples.

In the diagnostic industry, it is used to detect infections such as ocular infection, urinogenital infection, lung infection, respiratory viruses, neurotropic viral infection and tuberculosis infections.

HSV strains, EBV infection, VZV, CMV, T.gondii, influenza and adenoviral like pathogens can be detected with the help of the ready to use standard multiplex pathogen detection kits. For more details please read our previous article: microbial genetics.

the present method is utilized in the categorization and identification of pathogens.

It is also used in the multiple SNP genotyping.

More than single SNPs are encountered using the multiplex method. For example, thalassemia. In this blood-born inherited disease different SNPs such as IVS1-1, IVS1-5, IVS (G-C), CD5 and CD15 etc can be detected in a single reaction.

It is a very useful tool in genetically modified organism studies.

It is also useful in forensic studies.

In forensic studies, the different locus is targeted for identification of the organism, by using the multiplex PCR, multiple loci can be screened in a single experiment.

The multiplex PCR is helpful in mutation detection and polymorphism analysis.

Mutation detection even becomes very rapid and cost-effective, after the development of the mPCR. Y chromosome microdeletion is the best example of the application of multiplex PCR in mutation detection. More than 12 markers of the Y chromosome are used in the microdeletion studies. By combining all the markers in a single reaction helps to make the work easy. Multiplex PCR facilitates studies of multiple markers in a single reaction.  

Broda range DNA deletion studies can be possible by using the multiplex PCR.

It is used in linkage analysis studies.

Qualitative and quantitative analysis of template DNA is become possible by using multiplex PCR.

Multiplex PCR in combination with the real-time PCR is even more valuable and useful in the quantitative studies.

Real-time multiplex PCR is a great tool for template quantification. Furthermore, the quantitative analysis of multiple pathogens is also possible with the help of real-time multiplex PCR.

Importantly, One of the major factors in multiplex PCR is the length of the amplicon. Design the multiplex assay in such a way that each primer amplifies the template that produces the fragments between the length of 50 to 200bp (maximum).

If the product length is longer, the efficiency of the PCR reaction decreases.

Also, individual products are distinguishable from one other.   

Another advancement in the multiplex PCR is the use of real-time monitoring by the real-time PCR. By combining several reactions in one, the concentration of different pathogens and templates can be detected using the quantitative PCR.

Furthermore, the multiplex real-time PCR is faster and more accurate than the conventional PCR.

FACT: Multiplex PCR identifies 98% of deletion in case of DMD in all 19 exons. 


The multiplex PCR is more advantageous over the uniplex PCR. Therefore, the development of new multiplex PCR protocols for different disorders is essential, although, it is difficult to develop multiplex PCR assay, not impossible.

Yet, the multiplex PCR method is not a gold standard method for prenatal studies. More data and research are required to make it ready for prenatal studies.