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SF-qPCR: Strand Displacement-Based Fast Quantitative Polymerase Chain Reaction

SF-qPCR: Strand Displacement-Based Fast Quantitative Polymerase Chain Reaction Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid (N) gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25–40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioChip Journal Springer Journals

SF-qPCR: Strand Displacement-Based Fast Quantitative Polymerase Chain Reaction

Kim, Jiae; Jung, Cheulhee
BioChip Journal , Volume 16 (1) – Mar 1, 2022
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References (28)

Publisher
Springer Journals
Copyright
Copyright © The Korean BioChip Society 2022
ISSN
1976-0280
eISSN
2092-7843
DOI
10.1007/s13206-021-00044-x
Publisher site
See Article on Publisher Site

Abstract

Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid (N) gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25–40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis.

Journal

BioChip Journal – Springer Journals

Published: Mar 1, 2022

Keywords: Quantitative PCR; SF-qPCR; SD polymerase; Strand displacement activity; BRCA; SARS-CoV-2

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