When to Use a Multiplex PCR? Explained by 8 Real Applications – Genetic Education
When to Use a Multiplex PCR?

When to Use a Multiplex PCR? Explained by 8 Real Applications

“Multiplex PCR provides flexibility to multiplex the sample for many markers. Here is a list of 10 examples in which you can use a multiplex PCR approach.” 

PCR is a powerful technique used in molecular genetics that enables studying a specific DNA sequence or a gene. This way, genes and genetic disorders can be studied. Various PCR forms and variations have been adopted for specific applications. 

Multiplex PCR is a variant of PCR that can study multiple targets or markers in a single run. By doing so various alleles associated with a disease can be studied.

Multiplexing is a complex and error-prone process. It can result in false-positive and false-negative results. That we all know. So you may have a question, when to use a multiplex PCR or in which conditions it can be used?

Here in this article, I will provide you 8 real applications in which a multiplex PCR is used. Based on that I will give you a guide on when and in which conditions it is needed. 

Stay tuned. 

8 Applications of Multiplex PCR

Illustration of a multiplex PCR reaction
The image represents the multi-template multiplex PCR.

Forensic DNA Profiling:

One of the well-known classic applications of multiplex PCR is in forensic analysis and paternity testing. In this process, short tandem repeat (STR) markers are amplified and studied. These repetitive sequence numbers vary among individuals and are used for detection. 

Usually, 16, 21 or 25 different STR markers can be multiplexed and studied using the multiplex PCR. The results are analyzed using the capillary gel electrophoresis technique.

When your experiment needs STR or VNTR typing, instead of running each marker in a single reaction, you can use multiplex PCR. Note that ready-to-use kits are also available.

Related article:

HLA Typing:

Multiplex PCR is also extensively used in Human Leukocyte Antigen typing in the transplantation field. Organ donor and recipient compatibility can be investigated by amplifying various HLA genotypes in a single reaction. The present HLA typing method is rapid and accurate.  

HLA typing using multiplex PCR is comparatively easy to perform as various ready-to-use kits for quantitative analysis and fragment analysis is available in the market. When your research includes HLA typing or anything related to organ transplantation compatibility, you can use multiplexing. 

Pathogen Detection:

Yet another application of the present technique is in pathogen detection. Different pathogens and microbial strains can be detected in a single multiplex reaction. Thus, it can be used in fields like the metagenomic studies

Although it can not determine unknown pathogens or strains, if you are still doing some microbiology analysis or metagenomic experiments, you can consider multiplex PCR. 

Read more: Why Microbial Genetics Is The Future of Microbiology? Comparison and Applications.

Y chromosome microdeletion: 

Multiplex PCR is also extensively employed for Y chromosome microdeletion analysis. More than 300 different sequence-tagged sites are identified and used for microdeletion analysis associated with the Y chromosome

Ideally, 20 to 30 markers are used for diagnosis purposes. When you are doing research or setting up a diagnostic test for the Y chromosome, you can consider multiplexing. Note that various kits and peer-reviewed literature are available on this topic. 

Cancer mutation analysis:

A recent advancement in cancer research is multiplexing. Various cancer-associated mutations can be screened using the present approach. For instance, more than 100 known P53 mutations are associated with various types of cancer. 

It’s difficult to study these mutations one by one, although we have a high throughput NGS sequencing option available, multiplexing various mutations in a single PCR run saves time with increased sensitivity and accuracy. 

If you are doing research on a particular type of cancer, or various cancer-causing mutations, you can consider a multiplex PCR approach. 

Hereditary disease screening:

One potential application of multiplex PCR is in the screening of hereditary diseases. For example, Thalassemia, Huntington’s disease, etc. Multiple mutations associated with a disease can be multiplexed and screened in a single run. 

However, for disease diagnosis, multiplexing is not recommended. For research purposes, if you want to study “some” inherited genetic diseases, you can use multiplex PCR. 

Food safety testing:

The quantitative Multiplex PCR approach is further employed for food safety testing, in routine. Ready-to-use kits, assays and literature are available to detect foodborne pathogens and contaminants. 

If you are setting up a food safety lab, or doing research on some related topics, you can blindly trust the multiplex PCR approach to boost up your experiment. 

Plant research:

RAPD (Random Amplification of Polymorphic DNA) is a marker widely used in plant research and marker-assisted selection studies. The polymorphic nature of the RAPD marker is used to characterize and classify various plant species. 

If you are doing research in this field, you can also consider the RAPD multiplex approach. 

When not to use multiplex PCR? 

sNow, you also have to know when you can not use the multiplex PCR assay. Wrongly choosing multiplex PCR potentially increases the chances of false-positive and -negative results. Unwanted results cause non-specific amplification, primer-dimer and unknown target amplifications. 

Highly complex experiment: 

If there are one or more targets having chances of non-specific amplification and primer dimers, avoid multiplexing such templates. 

Low abundance targets:

Straight forward “No” for low abundance targets. If the target allele or gene is present in very low abundance or has fewer copies, avoid using them in any multiplex experiment. Here, the sensitivity is compromised and can not give desirable results. 

Repetitive DNA-rich and larger target:

If the target contains any larger repetitive block or larger amplicon size, avoid multiplexing such templates. Multiplexing these templates in a PCR, results in inadequate amplification. 

In addition, do not use multiplex PCR assay for 

  • Impure DNA or contaminated DNA.
  • Clinical or diagnosis testing. 
  • Unknown templates. 
  • Templates that are located very near. 
  • Templates with less amplicon size difference. 

Wrapping up

Multiplexing in PCR saves time with improved sensitivity and accuracy and is highly recommended if possible. However, expertise is required to study and understand the complex multiplexing results. 

Before proceeding with multiplexing, I highly advise conducting individual reactions for each target (uniplex or single reactions). Carefully analyzing these results will help determine whether they can be effectively combined into a multiplex assay or not.

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