Multiplex PCR Coupled with Fragment Analysis

A Hi-Plex Approach to Simultaneous Genomic Targets Detection

The introduction of modern-day PCR in 1985 revolutionized the field of genomics and had a profound impact on the understanding that scientists could gain at the DNA level of a cell or organism. As genetic analysis technologies have advanced, researchers are asking bolder questions spanning from basic cellular biology to applied and translational medicine. Across this gamut, the advantage to query and analyze a large number of genetic targets simultaneously to get to the answer, remains undisputable.

Standard PCR Coupled with Capillary Electrophoresis (CE)

While quantitative PCR is the mainstay for rapid and concurrent analysis of several genomic sequences, it can be limited by the number of unique fluorophores, and therefore, the number of genomic targets that can be queried. In situations where a high multiplexing capability is desired, standard PCR coupled with capillary electrophoresis offers a versatile solution to detect multiple species in a single reaction through a fragment analysis-based approach.

What is Fragment Analysis?

Fragment analysis is a genetic analysis method that can separate fluorescently-labeled genomic products from multiplexed PCR based on their size, with up to a 2 bp resolution difference. With a practical operating space between 100 nucleotides and 600 nucleotides, this could theoretically detect 250 distinct peaks – meaning targets – per fluor. Coupled with the multiple fluors that can be run in a single capillary, this enables a potential multiplexing capability of a large number of targets from a single sample. In addition, it may be possible to PCR amplify different samples with the same primers, but labelled with different sets of fluorophore-labeled dyes. By combining these amplicons from different samples before electrophoresis, this could allow up to 4 samples to be analyzed in a single capillary. Although a sample multiplexing approach would need to be fully optimized, it could be very powerful for detecting and distinguishing genomic targets efficiently across many different samples.

Related: Fragment Analysis Applications

Multiplex PCR with Fragment Analysis

Since the onset of the SARS-CoV-2 pandemic in 2020, there has been an increased interest in analytical tools that enable the simultaneous detection of various respiratory pathogens in a given sample. Given that the SARS-CoV-2 infection typically begins with common flu-like indications such as coughing and general malaise, characterization of the etiological agent of infection, therefore is a critical step to formulate the downstream approach.  A multiplex  PCR  with  fragment  analysis-based  approach  can  be  used  to detect  and  determine  the  causative  agent  with  high  sensitivity  and  specificity  by designing  primers  against  various  respiratory  pathogens  that  yield  unique  amplicon sizes after amplification.

Related: Application Note: Multiplex Detection of Respiratory Pathogens using Fragment Analysis by Capillary Electrophoresis

Fragment Analysis and Detection of Genetic Aberrations

Versatility for Genetic Target Identification Across Research Areas

In addition to its application in infectious diseases, fragment analysis has also been shown to be a powerful tool to determine genetic aberrations such as:

• Translocation for multiple rearrangements in cancer.
• Screen various mutations across a particular gene to determine the etiological cause of the disease.
• Identify genetic polymorphisms across several variants simultaneously to predict pharmacological response to drugs.

The versatility of this approach for genetic target identification is unparalleled and can be broadly applied to any research area where simultaneous detection of several targets, at the DNA or RNA level, is desirable.

Related

• • Sanger Sequencing and Fragment Analysis by Capillary Electrophoresis
  • Sequencing & Fragment Analysis Applications

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Citations

1. Algeciras-Schimnich, A., et al., Evaluation of the PAX8/PPARG translocation in follicular thyroid  cancer  with  a  4-color  reverse-transcription  PCR  assay  and  automated  high resolution fragment analysis. Clin Chem, 2010. 56(3): p. 391-8.
2. Furtado, L.V., et al., A multiplexed fragment analysis-based assay for detection of JAK2 exon 12 mutations. J Mol Diagn, 2013. 15(5): p. 592-9.
3. Bouvet, R., et al., PharmFrag: An Easy and Fast Multiplex Pharmacogenetics Assay to Simultaneously  Analyze  9  Genetic  Polymorphisms  Involved  in  Response  Variability  of Anticancer Drugs. Int J Mol Sci, 2020. 21(24).