{"id":16399,"date":"2022-05-23T03:09:23","date_gmt":"2022-05-23T03:09:23","guid":{"rendered":"https:\/\/admin.acceleratingscience.com\/behindthebench\/?p=16399"},"modified":"2022-05-23T03:09:23","modified_gmt":"2022-05-23T03:09:23","slug":"multiplex-pcr-coupled-with-fragment-analysis","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/multiplex-pcr-coupled-with-fragment-analysis\/","title":{"rendered":"Multiplex PCR Coupled with Fragment Analysis"},"content":{"rendered":"

A Hi-Plex Approach to Simultaneous Genomic Targets Detection<\/h2>\n

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.<\/p>\n

Standard PCR Coupled with Capillary Electrophoresis (CE)<\/h3>\n

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.<\/p>\n

What is Fragment Analysis?<\/h2>\n

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 \u2013 meaning targets \u2013 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.<\/p>\n

Related<\/strong>: Fragment Analysis Applications<\/a><\/p>\n

\"genetic<\/h2>\n

Multiplex PCR with Fragment Analysis<\/h2>\n

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.\u00a0 A multiplex\u00a0 PCR\u00a0 with\u00a0 fragment\u00a0 analysis-based\u00a0 approach\u00a0 can\u00a0 be\u00a0 used\u00a0 to detect\u00a0 and\u00a0 determine\u00a0 the\u00a0 causative\u00a0 agent\u00a0 with\u00a0 high\u00a0 sensitivity\u00a0 and\u00a0 specificity\u00a0 by designing\u00a0 primers\u00a0 against\u00a0 various\u00a0 respiratory\u00a0 pathogens\u00a0 that\u00a0 yield\u00a0 unique\u00a0 amplicon sizes after amplification.<\/p>\n

Related<\/strong>: Application Note: Multiplex Detection of Respiratory Pathogens using Fragment Analysis by Capillary Electrophoresis<\/a><\/p>\n

Fragment Analysis and Detection of Genetic Aberrations<\/h2>\n

Versatility for Genetic Target Identification Across Research Areas<\/h3>\n

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:<\/p>\n