Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2019-12

AUTHORS

Aditya Rajagopal, Dominic Yurk, Claudia Shin, Karen Menge, Lucien Jacky, Scott Fraser, Thomas A. Tombrello, Gregory J. Tsongalis

ABSTRACT

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity. More... »

PAGES

1053

References to SciGraph publications

  • 2015-12. Doubling Throughput of a Real-Time PCR in SCIENTIFIC REPORTS
  • 1996-03. Molecular Beacons: Probes that Fluoresce upon Hybridization in NATURE BIOTECHNOLOGY
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/s41598-018-37732-y

    DOI

    http://dx.doi.org/10.1038/s41598-018-37732-y

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1111775524

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/30705333


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