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

Kevin Maafu Juma,Yuri Ando,Mika Ishitani,Koichiro Suzuki,Itaru Yanagihara,Yukiko Nakura,Wakao Fukuda,Masaya Yamagata,Kiyoshi Yasukawa,Kaichi Hayashi,Shinsuke Fujiwara,Teisuke Takita,Kenji Kojima

doi:10.1007/s11033-021-07098-y

Abstract

BackgroundRecombinase (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.MethodsUntagged 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.ResultsThe minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 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.ConclusionsThe reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.Supplementary InformationThe online version of this article contains supplementary material available 10.1007/s11033-021-07098-y.

Highlights

Recombinase polymerase amplification (RPA) amplifies a target DNA sequence at a constant temperature around 37–42 °C [1,2,3]
We examined the effects of each component of the reaction solution on the RPA reaction efficiency and optimized the reaction conditions using a statistical method [5]
We examined the effects of N- and C-terminal His-tags of uvsY on its function in RPA using SARS-CoV-2 DNA as a model target

Summary

Introduction

Recombinase polymerase amplification (RPA) amplifies a target DNA sequence at a constant temperature around 37–42 °C [1,2,3]. Strand-displacing DNA polymerase (Pol) extends the primer while SSB binds to the dispatched strand. In this way, a new DNA strand is synthesized. 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. Results The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 1 05, 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, uvsYNhis, uvsY-Chis, and uvsY-NChis, respectively. Conclusions The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA

Methods

Results

Conclusion