Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.

Autor: Juma KM; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Takita T; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Yamagata M; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Ishitani M; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Hayashi K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Kojima K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.; Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo, 670-8524, Japan., Suzuki K; The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, 565-0871, Japan., Ando Y; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo, 669‑1337, Japan., Fukuda W; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo, 669‑1337, Japan., Fujiwara S; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo, 669‑1337, Japan., Nakura Y; Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi-shi, Osaka, 594-1101, Japan., Yanagihara I; Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi-shi, Osaka, 594-1101, Japan., Yasukawa K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan. yasukawa.kiyoshi.7v@kyoto-u.ac.jp.
Jazyk: angličtina
Zdroj: Molecular biology reports [Mol Biol Rep] 2022 Apr; Vol. 49 (4), pp. 2847-2856. Date of Electronic Publication: 2022 Jan 31.
DOI: 10.1007/s11033-021-07098-y
Abstrakt: 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.)
Databáze: MEDLINE
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