l icterohaemorrhagiae l canicola l grippotyphosa id li84 lc87 atcc 23604 o d  (ATCC)

Journal: PeerJ

Article Title: The concentration of single-stranded DNA-binding proteins is a critical factor in recombinase polymerase amplification (RPA), as revealed by insights from an open-source system

doi: 10.7717/peerj.19758

Figure Lengend Snippet: (A) Schematic representation of T4 HR D-loop. During this process, single-stranded DNA (ssDNA) is coated with gp32, while the mediator protein UvsY displaces gp32 from the ssDNA and facilitates the loading of the recombinase UvsX. The loading of UvsX promotes the formation of presynaptic filaments that search for homologous regions within large double-stranded DNA (dsDNA) and execute base pairing with the ssDNA. The 3′-OH of the ssDNA oligonucleotide serves as a primer for DNA polymerases. (Panel created with BioRender.com). (B) A total of 12.5% SDS-PAGE of purified T4 HR proteins is presented. The gel demonstrates the purity and molecular weight of gp32, UvsX, and UvsY, with their relative migration indicated by arrows. (C) D-loop assembly mediated by T4 HR components was compared to that of bacterial RecA. Bacterial RecA, at a concentration of 300 nM, annealed a Cy5-labeled DNA substrate at two nM to a complementary region of a supercoiled pGEM plasmid in the presence of 5 mM ATP (lanes 2 and 3). The Cy5-labeled DNA substrate was unable to self-anneal to the supercoiled template (lanes 4 and 5). D-loop formation was assessed using two concentrations of the individual components of the T4 HR system in the presence of ATP (lanes 4 to 11). Reactions containing gp32, UvsX, and UvsY were performed at concentrations of 600 nM (lanes 4, 6, 8) or six µM (lanes 5, 7, 9, 10, 11, 12).

Article Snippet: The plasmids encoding the protein products used in this study have been deposited in Addgene as follows: pET19b-pss-gp32 (Plasmid #236044), pET19b-pps-UvsY (Plasmid #236045), pET19b-pps-UvsX (Plasmid #236046), pCOLDI-KF-BstDNApolI (Plasmid #236047), and pET19b-pps-EndoIV from Thermus thermophilus (Plasmid #236048).

Techniques: SDS Page, Purification, Molecular Weight, Migration, Concentration Assay, Labeling, Plasmid Preparation

Journal: PeerJ

Article Title: The concentration of single-stranded DNA-binding proteins is a critical factor in recombinase polymerase amplification (RPA), as revealed by insights from an open-source system

doi: 10.7717/peerj.19758

Figure Lengend Snippet: (A) Diagram indicating the synthetic SARS-CoV-2 DNA cloned in a pET28b plasmid and the number of amplified base pairs at a specific pair of RPA oligonucleotide would amplify for each gene. The diagram also depicts the RNA promoter sequences that facilitate the generation of SARS-CoV-2-like transcripts. (B) PCR amplification results for the P, E, and N genes utilizing both free oligonucleotides and 5′-biotinylated oligonucleotides. (C) RPA reactions targeting the P, E, and N genes were conducted both in the absence and presence of 40 mM potassium acetate, using 5′-OH and 5′-biotinylated oligonucleotides. The amplified RPA products are marked with a red asterisk. (D) Assessment of the impact of varying concentrations of T4-HR proteins on RPA efficiency. RPA reactions were performed with increasing concentrations of UvsY (0, 0.47, 0.94, 1.88, 2.82, 3.76 µM), UvsX (0, 0.6, 1.35, 2.7, 4, 5.4 µM), and gp32 proteins (0, 3.33, 6.6, 13.33, 20, 26.6 µM). The relative migration of the RPA products and primer-dimers is indicated by arrows. In each standard RPA reaction, the concentration of one component of the T4-HR system was increased while maintaining the other two proteins at constant levels.

Article Snippet: The plasmids encoding the protein products used in this study have been deposited in Addgene as follows: pET19b-pss-gp32 (Plasmid #236044), pET19b-pps-UvsY (Plasmid #236045), pET19b-pps-UvsX (Plasmid #236046), pCOLDI-KF-BstDNApolI (Plasmid #236047), and pET19b-pps-EndoIV from Thermus thermophilus (Plasmid #236048).

Techniques: Clone Assay, Plasmid Preparation, Amplification, Migration, Concentration Assay