Publication detail

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

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"
}