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#34166918   2021/06/10 To Up

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.
Kevin Maafu Juma, Teisuke Takita, Kenji Ito, Masaya Yamagata, Shihomi Akagi, Emi Arikawa, Kenji Kojima, Manish Biyani, Shinsuke Fujiwara, Yukiko Nakura, Itaru Yanagihara, Kiyoshi Yasukawa

1558 related Products with: Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

250tests200ug100tests250Tests200ul100 mg200ul96 tests100tests

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#33070788   2020/10/19 To Up

p67: a cryptic lysosomal hydrolase in ?

p67 is a type I transmembrane glycoprotein of the terminal lysosome of African trypanosomes. Its biosynthesis involves transport of an initial gp100 ER precursor to the lysosome, followed by cleavage to N-terminal (gp32) and C-terminal (gp42) subunits that remain non-covalently associated. p67 knockdown is lethal, but the only overt phenotype is an enlarged lysosome (~250 to >1000 nm). Orthologues have been characterized in Dictyostelium and mammals. These have processing pathways similar to p67, and are thought to have phospholipase B-like (PLBL) activity. The mouse PLBD2 crystal structure revealed that the PLBLs represent a subgroup of the larger N-terminal nucleophile (NTN) superfamily, all of which are hydrolases. NTNs activate by internal autocleavage mediated by a nucleophilic residue, i.e. Cys, Ser or Thr, on the upstream peptide bond to form N-terminal α (gp32) and C-terminal β (gp42) subunits that remain non-covalently associated. The N-terminal residue of the β subunit is then catalytic in subsequent hydrolysis reactions. All PLBLs have a conserved Cys/Ser dipeptide at the α/β junction (Cys241/Ser242 in p67), mutation of which renders p67 non-functional in RNAi rescue assays. p67 orthologues are found in many clades of parasitic protozoa, thus p67 is the founding member of a group of hydrolases that likely play a role broadly in the pathogenesis of parasitic infections.
Carolina M Koeller, Terry K Smith, Andrew M Gulick, James D Bangs

1975 related Products with: p67: a cryptic lysosomal hydrolase in ?

96T100 μg100ug Lyophilized100 μg100ug Lyophilized100 μg1 Set100 μg1 Set96 samples

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#31254238   2019/06/28 To Up

Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.
Ruopeng Cai, Zijing Wang, Gang Wang, Hao Zhang, Mengjun Cheng, Zhimin Guo, Yalu Ji, Hengyu Xi, Xinwu Wang, Yibing Xue, Sadeeq Ur Rahman, Changjiang Sun, Xin Feng, Liancheng Lei, Yigang Tong, Wenyu Han, Jingmin Gu

2242 related Products with: Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein.

1000 TESTS/0.65ml100ug100ug100ug1 Set100ug100ug1 mg100ug Lyophilized1 module100ml

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#30426974   // To Up

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification.

Nucleic acid amplification has permeated every field in the life sciences since the introduction of the classic polymerase chain reaction (PCR) method in 1983. Yet, despite its fundamental reach, PCR has been constrained within the walls of a laboratory, due to its requirement for a sophisticated thermocycling machine, limiting external application in low-resource settings. New isothermal amplification strategies are seeking to break through traditional laboratory boundaries by providing nucleic acid replication at constant temperatures. Of these methods, recombinase polymerase amplification (RPA) is one of the fastest developing, experiencing rapid uptake and market, even though it was introduced comparatively late. Critically, RPA's technology potentiates highly accessible and sensitive nucleic acid amplification outside of laboratory, and even self-testing. Here we provide a comprehensive review of the equipment-free simplicity of RPA over its first decade of development. Our review includes key knowledge of RPA technology, such as its reaction components, mechanism, sensitivities and specificities, and distinctive detection methods. The review also provides know-how for developing RPA assays, and information about commercially available RPA reaction kits and accessories. We summarise critical RPA experimental tips and issues available through data mining the published literature, to assist researchers in mastering the RPA reaction. We also outline influential hotspots of RPA development, and conclude with outlooks for future development and implications for eclipsing PCR and further revolutionising the life sciences.
Jia Li, Joanne Macdonald, Felix von Stetten

2426 related Products with: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification.

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#29634784   2018/04/10 To Up

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292-296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.
Kiran Pant, Brian Anderson, Hendrik Perdana, Matthew A Malinowski, Aye T Win, Christopher Pabst, Mark C Williams, Richard L Karpel

2143 related Products with: The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

1000 TESTS/0.65ml5 x 200 ug50 100.00 ug1mg5 x 200 ug96tests200 units50 100.00 ug

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#29292222   2017/12/30 To Up

ssDNA hybridization facilitated by T7 ssDNA binding protein (gp2.5) rapidly initiates from the strand terminus or internally followed by a slow zippering step.

The hybridization of two complementary ssDNA is essential for molecular biology and biological physics. T7 ssDNA binding protein (gp2.5) can rapidly facilitate this hybridization, but the mechanism and kinetic process remain unknown. As determined by fluorescence resonance energy transfer (FRET) and rapid kinetic analysis methods, gp2.5 binding coiling ssDNA to form gp2.5-ssDNA complex is the rate-limiting step for the entire DNA hybridization process. Afterward, the hybridization initiates from either the terminus or the internal part of ssDNA at a nucleation rate of 0.45 s. The remaining DNA strands are hybridized in a zippering mode at a rate of 0.07 s, limited by the dissociation of gp2.5 from ssDNA. The gp2.5-faciliated hybridization rate constant (74 μMs) is much higher than the spontaneous hybridization rate (1.7 μMs) in the absence of gp2.5. These hybridization mechanism and kinetic process are different from those by other ssDNA binding proteins, such as T4 gp32, Escherichia coli SSB protein and recA protein. Compare with E. coli SSB, the relatively slower association of gp2.5 to ssDNA and faster dissociation of gp2.5 from ssDNA are probably the major reasons to facilitate the rapid hybridization. This study provides valuable insights into the molecular mechanism of the recombination and repair processes and proposes a basis for improvement in their associated biotechnologies.
Zhenyu Zou, Siying Wu, Jingyuan Xiong, Huangyuan Li, Yiguo Jiang, Huidong Zhang

2580 related Products with: ssDNA hybridization facilitated by T7 ssDNA binding protein (gp2.5) rapidly initiates from the strand terminus or internally followed by a slow zippering step.

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#28819114   2017/08/17 To Up

Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein.

Isothermal amplification methods for detection of DNA and RNA targets have expanded significantly in recent years, promising a new wave of simple and rapid molecular diagnostics. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products. Here we report the amplification of discrete target fragments of several kilobases at 37 °C from both double- and single-stranded circular template DNA using specific primer pairs. In contrast to existing methods, this amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. In addition to the discrete amplicon products, this method also produces higher molecular weight products consisting of multiple repeated copies of the amplicon and template DNA. We demonstrate that two features of gp32 enable this amplification: a facilitation of primer strand invasion into double-stranded DNA, and a suppression of non-homologous primer annealing and nonspecific amplification. The ability presented here to produce long, discrete DNA products in an isothermal reaction extends the scope of isothermal amplification to enable more useful applications of these promising methods.
Yinhua Zhang, Nathan A Tanner

2279 related Products with: Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein.

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#28416680   2017/04/17 To Up

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 10 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.
Carey Phelps, Brett Israels, Davis Jose, Morgan C Marsh, Peter H von Hippel, Andrew H Marcus

1371 related Products with: Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.

10 200 ug200 ug100 ug100 ug5 x 200 ug5 x 200 ug100.00 ug1mg 100ul200 100 µg

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#27974172   2016/12/06 To Up

Advances in structural studies of recombination mediator proteins.

Recombination mediator proteins (RMPs) are critical for genome integrity in all organisms. They include phage UvsY, prokaryotic RecF, -O, -R (RecFOR) and eukaryotic Rad52, Breast Cancer susceptibility 2 (BRCA2) and Partner and localizer of BRCA2 (PALB2) proteins. BRCA2 and PALB2 are tumor suppressors implicated in cancer. RMPs regulate binding of RecA-like recombinases to sites of DNA damage to initiate the most efficient non-mutagenic repair of broken chromosome and other deleterious DNA lesions. Mechanistically, RMPs stimulate a single-stranded DNA (ssDNA) hand-off from ssDNA binding proteins (ssbs) such as gp32, SSB and RPA, to recombinases, activating DNA repair only at the time and site of the damage event. This review summarizes structural studies of RMPs and their implications for understanding mechanism and function. Comparative analysis of RMPs is complicated due to their convergent evolution. In contrast to the evolutionary conserved ssbs and recombinases, RMPs are extremely diverse in sequence and structure. Structural studies are particularly important in such cases to reveal common features of the entire family and specific features of regulatory mechanisms for each member. All RMPs are characterized by specific DNA-binding domains and include variable protein interaction motifs. The complexity of such RMPs corresponds to the ever-growing number of DNA metabolism events they participate in under normal and pathological conditions and requires additional comprehensive structure-functional studies.
S Korolev

2592 related Products with: Advances in structural studies of recombination mediator proteins.

1001 mL5050100500 1mg1010001021. Set

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