Amplifying different DNA sequences or different DNA templets by using the different set of primers in a single PCR reaction is refers to as a multiplex PCR.

PCR technology is now a routine day method used in the diagnostic industries. Because it is efficient and accurate. However, the cost of the experiment and the time utilised in performing the experiment creates a major set back for the technology.

The cost of a single PCR experiment is very high. Also, the entire procedure takes more than 4 hours for a single experiment, starting from DNA extraction to the gel electrophoresis.

We can not minimise the time and the cost of the experiment but what if we perform multiple experiments at the same time and using the same quantity of reagents?

It saves money and time, right.

Multiplex PCR facilitates multiple reactions in one single run which means we can analyse multiple template locus in 4 hours (time required for a single experiment).

The technique is first described in the year 1988 by Jeffrey S. Chamberlain and coworkers. They had used the multiplex PCR assay to screen the Duchenne muscular dystrophy locus. 

They had demonstrated the technique is for preferentially for prenatal and postnatal diagnosis. They had used multiple sets of primers for encountering multiple deletions in the dystrophin gene.

The multiplex PCR reaction is a technique widely used in virology and in the single gene 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 present into the templet DNA or sequences of multiple temples in a single PCR reaction.”

Here, in the multiplexing we are using different sets of primer, therefore we need more amount of the PCR reagents such as Taq DNA polymerase, dNTPs and PCR buffer, but that’s not the case always.  

In simple words, we can say it is the combination of more than two PCR reactions, hence it can save the time of the reaction and more mutations can be screened in a single reaction.

Based on the type of the template 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 reaction of PCR. For that, different sets of primers are used.

However, it does not mean to pool different samples in a single reaction.

It is used if the sample or the organism is infected by the multiple pathogens originated infection. We can say, it is applicable in the detection of different strains or species of pathogens.

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.

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

How to develop a multiplex PCR assay?

Developing an MPCR assay is not as easy as the uniplex-PCR because it required high expertise and trial and error experiment runs for one particular multiplex reaction.

Obviously, more targets in the DNA sequence which means we required more amount of PCR reagents.

If we are designing a single templet multiplex PCR assay, use high amount or higher concentration of templet DNA. The concentration of the templet DNA should be more than it is used for the single reaction PCR.

Simultaneously, the Taq DNA concentration and the MgCl2 concentration must be also higher for better performance.

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 each other. One set of the primer can not cross bind with another set of primer. Further, it can not form dimers.  

This means that each primer set must be unique and precious.

The GC content of the primers are between 45% to 60% and the melting temperature must be between 55°C to 60°C.

Ideally, the annealing temperature of all the primer sets is very nearer to one another (the difference of annealing temperature is not more than 4°C).

Furthermore, the length of each primer can fail the reaction badly, hence the ideal length of each set of primer should be in between 18 to 30. Ideally, it can be between 20 to 22 nucleotides.

Read our PCR primer design guidelines:

PCR primer design guidelines

The PCR reaction cycles also play a vital role in achieving amplification for each template DNA. In some cases, if the PCR cycles are more, the amplification cannot be properly achieved because of the unavailability of the PCR reagents or templates in later cycles.

Use proper cycling condition, not more or not less and this should be achieved by practising and experimenting repeatedly.

25 to 30 cycles are sufficient for it.

Now, our multiplex PCR assay is ready to perform.

Protocol for multiplex PCR:

The protocol for multiplex reaction varies from the experiment to experiments. For example, the protocol used in the Y chromosome microdeletion multiplex PCR is almost the same as the uniplex PCR for every single marker for Y chromosome microdeletion. While the protocol for the DMD gene is more complex, required more amount of reagents and expertise.

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 avoid the non-specific bindings, primer dimer formation, hairpin formation and unavailability of the templates different components such as MgCl2, KCl, albumin, DMSO, betaine or Taq DNA polymerase can be added to the reaction, However, it depends on the results, what kind of problem is arise in the reaction.

Read more,

DNA sequencing Gene editing

Advantages of multiplex PCR:

  • The multiplex PCR assay consumes less time and manpower with greater efficiency.
  • The method is actually rapid and accurate.
  • By comparing different amplicons of a single template we can determine the quality of the template.
  • We can gather more information in a single run hence by utilizing less sample.
  • Also, the chance of pipetting errors is less in multiplex PCR.
  • Fewer consumables and chemicals used in the multiplex PCR.

 Limitations of multiplex PCR:

  • Although the technique is advantageous, the multiplexing is not applicable to all types of reaction.
  • For the larger amplicon such as 800bp or 1000bp, multiplex PCR might not work efficiently always.

Application of multiplex PCR:

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

In modern day science, the greatest application of PCR technology is in the study and identification of pathogen detection and virology.

The traditional microbiology culture technique is tedious and time-consuming, also, the chance of the contamination is always very high in the culture method.

The PCR allows pathogen detection in rapid time and with high precision. Furthermore, after the development of the multiplex PCR, the conventional PCR method is totally revolutionalised. Using the multiplexing method scientist can identify more than one pathogen or more than one strain of pathogen.

In a single reaction, multiple infections can be encountered using the multiplex PCR.

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

Pathogen identification is possible using multiplex PCR as well. Categorization of different viruses is possible by using this technique, we can group different pathogens and also, we can identify their strains.

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 marker 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.

Linkage analysis and RNA detection are further possible with the help of multiplex PCR.

Qualitative and quantitative analysis of template DNA is possible 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 amplify 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, 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.