Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.
Mol Biol Rep
; 49(4): 2847-2856, 2022 Apr.
Article
in English
| MEDLINE | ID: covidwho-1661716
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 × 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.CONCLUSIONS:
The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Viral Proteins
/
DNA, Viral
/
Bacteriophage T4
/
Nucleic Acid Amplification Techniques
/
DNA-Binding Proteins
/
SARS-CoV-2
/
Membrane Proteins
Language:
English
Journal:
Mol Biol Rep
Year:
2022
Document Type:
Article
Affiliation country:
S11033-021-07098-y
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