1. Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.
- Author
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Juma KM, Takita T, Yamagata M, Ishitani M, Hayashi K, Kojima K, Suzuki K, Ando Y, Fukuda W, Fujiwara S, Nakura Y, Yanagihara I, and Yasukawa K
- Subjects
- DNA, Viral genetics, SARS-CoV-2 genetics, Bacteriophage T4 enzymology, DNA, Viral chemistry, DNA-Binding Proteins chemistry, Membrane Proteins chemistry, Nucleic Acid Amplification Techniques, SARS-CoV-2 chemistry, Viral Proteins chemistry
- Abstract
Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA., Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels., Results: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 10
5 , 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs., Conclusions: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)- Published
- 2022
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