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

Rajagopal, Aditya and Yurk, Dominic and Shin, Claudia and Menge, Karen and Jacky, Lucien and Fraser, Scott and Tombrello, Thomas A. and Tsongalis, Gregory J. (2019) Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation. Scientific Reports, 9 . Art. No. 1053. ISSN 2045-2322. PMCID PMC6355831.

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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.

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Fraser, Scott0000-0002-5377-0223
Additional Information:© The Author(s) 2019. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Received: 18 January 2017. Accepted: 12 December 2018. Published: 31 January 2019. The authors gratefully acknowledge the financial support from the National Science Foundation Graduate Research Fellowship Program as well as Mr. Fred Farina, Dr. Mary Beth Campbell and the Caltech Office of Technology Transfer’s Rothenberg Innovation Grant. The authors would also like to thank Dr. David Baltimore at Caltech for critical advice and mentorship and Dr. Alaina Brinley for editing and proofreading the manuscript. Author Contributions: A.R. designed the experiments. A.R., D.Y., C.S., K.M. and L.J. performed the experiments. A.R., D.Y., S.F. and T.A.T. analyzed all data generated by these experiments. A.R., D.Y. and G.J.T. wrote the main manuscript. A.R. and D.Y. prepared all figures and tables. Competing Interests: This technology has been licensed by Caltech to a new startup company, ChromaCode, which is using the method to design tests for infectious diseases and oncology. A.R., D.Y., K.M., L.J. and G.J.T. hold equity in this startup. A.R., D.Y., K.M. and L.J. are currently employed by ChromaCode. G.J.T. is on the scientific advisory board for ChromaCode.
Funding AgencyGrant Number
NSF Graduate Research FellowshipUNSPECIFIED
Rothenberg Innovation Initiative (RI2)UNSPECIFIED
PubMed Central ID:PMC6355831
Record Number:CaltechAUTHORS:20190131-104830660
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92543
Deposited By: George Porter
Deposited On:31 Jan 2019 23:25
Last Modified:03 Oct 2019 20:45

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