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

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 co

SUBMITTER: Juma KM 

PROVIDER: S-EPMC8801280 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature