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ZHANG, H. YAN, Z. WANG, X. GAŇOVÁ, M. CHANG, H. LAŠŠÁKOVÁ, S. NEUŽIL, P. KORABEČNÁ, M.
Original Title
Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
Type
journal article in Web of Science
Language
English
Original Abstract
Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.
Keywords
real-time PCR; quantitative PCR; high-throughput; amplification
Authors
ZHANG, H.; YAN, Z.; WANG, X.; GAŇOVÁ, M.; CHANG, H.; LAŠŠÁKOVÁ, S.; NEUŽIL, P.; KORABEČNÁ, M.
Released
31. 8. 2021
Publisher
American Chemical Society
Location
WASHINGTON
ISBN
2470-1343
Periodical
ACS OMEGA
Year of study
6
Number
34
State
United States of America
Pages from
22292
Pages to
22300
Pages count
9
URL
https://pubs.acs.org/doi/10.1021/acsomega.1c02971
Full text in the Digital Library
http://hdl.handle.net/11012/203053
BibTex
@article{BUT172879, author="Haoqing {Zhang} and Zhiqiang {Yan} and Xinlu {Wang} and Martina {Gaňová} and Honglong {Chang} and Soňa {Laššáková} and Pavel {Neužil} and Marie {Korabečná}", title="Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel", journal="ACS OMEGA", year="2021", volume="6", number="34", pages="22292--22300", doi="10.1021/acsomega.1c02971", issn="2470-1343", url="https://pubs.acs.org/doi/10.1021/acsomega.1c02971" }