Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: Production and purity of PolI_G20c and ExnV1. ( a ) Molecular-weight and purity assessment of DNA polymerase I from Thermus phage G20c (PolI_G20c). Lane M, Precision Plus Protein Dual Colour Standards (Bio-Rad) molecular-mass marker; lane PolI_G20c, recombinant PolI_G20c purified by IMAC. ( b ) Production, purity and molecular-weight assessment of ExnV1. Lane M, PageRuler Unstained Broad Range Protein Ladder (Thermo Fisher Scientific); lane −, non-induced soluble crude extract of the ExnV1 expression culture; lane +, induced soluble crude extract of the ExnV1 expression culture; lane P, recombinant ExnV1 purified by IMAC.

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques: Molecular Weight, Marker, Recombinant, Purification, Expressing

Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: Determination of the activity of PolI_G20c. ( a ) Detection of polymerase activity of PolI_G20c at temperatures ranging from 20 to 85°C and comparison with the activity of IsoPol BST + and GoTaq polymerase. ( b ) Detection of 3′–5′ exonuclease activity of PolI_G20c and comparison with the activity of Phusion High-Fidelity polymerase exonuclease.

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques: Activity Assay, Comparison

Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: Evolutionary trees displaying the relationships ( a ) of PolI_G20c to PolI_Tth15-6 and deposited sequences encoding DNA polymerases I from thermophilic bacteriophages and ( b ) of ExnV1 to the 3′–5′ exonuclease domain in the closest related DNA polymerase I sequences. The trees are presented to scale, with branch lengths measured in number of substitutions per site. The trees were prepared with MEGA version X and were graphically edited with iTOL and Microsoft PowerPoint .

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques:

Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: Secondary structure of the DNA polymerase I PolI_G20c. The image was prepared in CCP 4 mg with PolI_G20c chain A (PDB entry 7r0k ).

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques:

Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: ( a ) The Klenow fragment (KF; PDB entry 1d9f ; chain A in brown) is superposed on PolI_G20c (PDB entry 7r0k ; chain A in ice blue) displayed as tubes. The black line indicates the border between the small and large domain as described by Ollis et al. (1985 ) and the orange line indicates where the larger, palm domain starts at residue 299 in PolI_G20c and residue 228 in KF (corresponding to residue 551 in full-length DNA polymerase I from E. coli ) according to InterPro. ( b ) Superposition of the best-matching structures based on a search using the DALI server. Chain A of DNA polymerase I from G. stearothermophilus (BF; PDB entry 1knc , gold), KF (PDB entry 2kzz , orange) and Taq polymerase (PDB entry 5ytf , violet) are superposed on chain A of PolI_G20c (ice blue) as ribbons. The structures match very well; however, α-helices 7 and 8 in PolI_G20c differ from all of the other compared enzymes and are part of a novel motif. The images were prepared with CCP 4 mg .

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques: Residue

Journal: Acta Crystallographica. Section D, Structural Biology

Article Title: Crystal structure of DNA polymerase I from Thermus phage G20c

doi: 10.1107/S2059798322009895

Figure Lengend Snippet: Comparison of the 3′–5′ exonuclease domain of ExnV1, PolI_G20c and the Klenow fragment.( a ) Residues 339–548 of the Klenow fragment (PDB entry 2kzz , orange) are superposed on the corresponding residues of PolI_G20c (PDB entry 7r0k , ice blue) and ExnV1 (PDB entry 7r0t , gold). The SβαR region of ExnV1 and PolI_G20c is marked with a red ring and includes α-helices 7 and 8 and β-strands 8, 9 and 10 of PolI_G20c, which are marked with arrows. ( b ) Sequence alignment of ExnV1, PolI_G20c and DNA polymerase I from E. coli generated in Jalview with conserved residues (threshold 100%) coloured according to ClustalX . The 3′–5′ exonuclease domain as defined in InterPro is underlined with a red dotted line and the SβαR structural motif is marked with a black box, including α-helices and β-strands of PolI_G20c, which are marked with red and blue lines, respectively, above the alignment. Crucial catalytic residues are marked with red boxes, important residues with secondary roles are marked with orange boxes and Glu357 is marked with a blue box. ( c ) As ( a ) but from another angle and with a box indicating an enlarged area containing crucial catalytic residues shown in ball-and-stick representation and important residues with secondary roles, including Glu357, displayed with fat bonds, where residues of the Klenow fragment, PolI_G20c and ExnV1 are coloured brown, blue and dark gold, respectively. ( d ) An enlarged area of ( c ). ( e ) The direct contact between the metal ions and catalytic residues of ExnV1 (numbered according to the corresponding residues of the Klenow fragment). ( f ) As ( c ) but with the metal ions and substrate from the model of the Klenow fragment displayed in light blue and the nucleotide and metal ions from ExnV1 displayed in violet.

Article Snippet: The temperature profile of PolI_G20c was compared with the thermoactivity of IsoPol BST + DNA polymerase (ArcticZymes Technologies) and of GoTaq DNA polymerase (Promega).

Techniques: Comparison, Sequencing, Generated

Journal: BMC Microbiology

Article Title: One-pot, uracil-DNA-glycosylase-aided, and multi-indicator nanobiosensor detection platform for identifying Brucella melitensis from the Brucella spp

doi: 10.1186/s12866-025-04189-9

Figure Lengend Snippet: Mechanism diagram of the mMCUDA protocol. The mMCUDA assay mainly includes UDGase-mediated cleavage action that followed dUTP-assisted MCDA amplification. Briefly, the UDGase-mediated cleavage for potential carryover contamination caused by mMCDA amplicons carrying dUTP was performed at 37 °C for 10 min after the premixed reaction mixtures (A and B, step 1). Then, the target fragments were exponentially amplified under the action of Bst DNA polymerase with strand displacement activity and ten MCDA primers capable of recognizing and binding to complementary regions on the target. Here, dTTPs in the dNTP mixtures are wholly replaced by dUTPs to ensure that produced amplicons carry uracil sites recognized by UDGase (A and B, steps 2–5). Finally, the FAM/Dig-CP1* and biotin-D1* primers were extended by binding to the target regions to yield abundant duplex FAM/Dig-amplicons-biotin complexes that can be captured by anti-FITC/anti-Dig antibodies (A and B, step 6). FAM, 6-carboxyfluorescein; mMCUDA, m ultiplex M CDA C ombined with dUTP-aided U DGase and D etector-based A uNPs-LFA biosensor; MCDA, multiple cross-displacement amplification; AuNPs-LFA, gold nanoparticles-based lateral flow assay

Article Snippet: Bst 6.0 DNA polymerase (stored in 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5)), 10 × reaction buffer (200 mM Tris-HCl, 500 mM KCl, 100 mM (NH4) 2 S0 4 , 20 mM MgS0 4 , 1% Tween-20 (pH 8.8)), Uracil-DNA Glycosylase (Heat-labile, Bacterium), and 100 mM MgSO 4 (Beyotime Biotechnology Co., Ltd., Shanghai, China).

Techniques: Amplification, Activity Assay, Binding Assay, Produced, Lateral Flow Assay

Journal: Journal of Clinical Microbiology

Article Title: Ultrasensitive and highly specific detection of the Brucella genus and B . melitensis by CRISPR/Cas12b‐multiple cross displacement amplification technique

doi: 10.1128/jcm.01532-24

Figure Lengend Snippet: Schematic illustration of CRISPR/Cas12b-based multiplex cross displacement amplification detection assay (CRISPR-MCDA-LFB). ( A ) A description of the principle of MCDA pre-amplification. ( B ) The trans-cleavage mechanism of Cas12b-gRNA (guide RNA) complex. ( C ) Outline of the CRISPR-MCDA-LFB assay workflow, including extraction of the DNA template, MCDA pre-amplification, the trans-cleavage of CRISPR-Cas12b, and the final verification of the reaction results with LFB or RTF instrument.

Article Snippet: The amplification reagent (10 × reaction buffer, dATP, dUTP, dGTP, dCTP, MgSO 4 , Bst 8.0 DNA polymerase) for MCDA and AUDG were provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China).

Techniques: CRISPR, Multiplex Assay, Amplification, Detection Assay, Extraction

Journal: Journal of Clinical Microbiology

Article Title: Ultrasensitive and highly specific detection of the Brucella genus and B . melitensis by CRISPR/Cas12b‐multiple cross displacement amplification technique

doi: 10.1128/jcm.01532-24

Figure Lengend Snippet: Confirmation tests for MCDA- Bcsp31 pre-amplification and CRISPR-MCDA-LFB assays. There were two main steps, including the verification of the MCDA pre-amplification products ( B–D ) and the confirmation of the trans-cleavage detection ( A, E, and F ). MCDA amplicons were verified by real-time turbidity ( B ), 1.5% agarose gel electrophoresis ( C ), and MG visual indicator ( D ). The Cas12b/gRNA-mediated trans-cleavage was then confirmed by real-time fluorescence analysis ( A ), UV visualization detection ( E ), and LFB ( F ). No. 1 was positive and No. 2 was negative. CRISPR, clustered regularly interspaced short palindromic repeats; MCDA, multiple cross displacement amplification. MG, malachite green; CRISPR/Cas12b, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12b protein; MCDA, multiple cross displacement amplification; gRNA, guide RNA; LFB, nanoparticle-based lateral flow biosensor.

Article Snippet: The amplification reagent (10 × reaction buffer, dATP, dUTP, dGTP, dCTP, MgSO 4 , Bst 8.0 DNA polymerase) for MCDA and AUDG were provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China).

Techniques: Amplification, CRISPR, Agarose Gel Electrophoresis, Fluorescence