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The widely used ET recombination requires an ssDNA product degraded by Rac phage protein E588 from dsDNA for strand invasion. However, proof of the ssDNA product is still elusive. The study provided three levels of proof sequentially. The probable ssDNAs degraded by E588 from the fluorescent plus-, minus-, or double-stranded dsDNA pET28a-xylanase exhibited a half fluorescence intensity of the corresponding dsDNAs, equivalent to the E588 degradation nucleotides half that of the total nucleotides degraded from the corresponding dsDNA. The ssDNA product degraded by E588 from the fluorescent minus-stranded dsDNA was confirmed by gradient gel-electrophoresis and two nuclease degradation reactions. Degraded by E588 from the dsDNA pET28a-xylanase that had a phosphorothioated plus-stranded 5'-terminus, the plus-stranded ssDNA product was separated via gel electrophoresis and recovered via a DNAclean kit. The recovered ssDNA product was proven to have intact 5'- and 3'-ends by DNA sequencing analysis. This study provides a solid foundation for the mechanism of ssDNA invasion.
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http://dx.doi.org/10.1016/j.bbrep.2024.101750 | DOI Listing |
Anal Chem
March 2025
Shandong Key Laboratory of Biophysics, Institute of Biophysics, Institute of Rural Revitalization, School of Pharmacy, Dezhou University, 253023 Dezhou, China.
Peripheral blood circulating tumor DNA (ctDNA) is a crucial liquid biopsy biomarker that correlates overall systemic tumor burden with malignant progression. However, identifying multiple single nucleotide polymorphisms (SNPs) in ctDNA presents significant challenges. In this study, we developed a rolling circle amplification (RCA)-supported multipedal DNA walker integrated with toehold-mediated strand displacement (TSDR) to facilitate the detection of ctDNA SNPs.
View Article and Find Full Text PDFOpen Biol
March 2025
Department of Pharmacology, First Faculty of Medicine, Charles University, Praha, Czech Republic.
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple.
View Article and Find Full Text PDFMikrochim Acta
March 2025
School of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, P. R. China.
The phosphorylation of nucleic acids mediated by 5'-polynucleotide kinase (PNK) exerts a crucial regulatory function in a wide range of significant cellular activities. Nevertheless, the current approaches for detecting PNK require expensive labeled probes and complex instrumentation, making it impossible to achieve real-time, on-site, and rapid analysis. Here, we take T4 PNK as a model and establish a novel colorimetric strategy for the detection of PNK activity and its inhibition by means of a coupled enzyme-assisted cyclic strand displacement amplification (SDA) and peptide nucleic acid (PNA)-gold nanoparticle (AuNP) based platform.
View Article and Find Full Text PDFHortic Res
March 2025
State Key Laboratory of Vegetable Biobreeding, Beijing Vegetable Research Center (BVRC), Beijing Academy of Agriculture and Forestry Science (BAAFS), No. 50, Zhanghua Road, Haidian District, Beijing 100097, China.
Pathogens significantly restrict the production of ( L. ssp. Pekinensis), with climate change and evolving planting patterns exacerbating disease prevalence.
View Article and Find Full Text PDFChem Bio Eng
February 2025
Department of Molecular Discovery, R&D, Novozymes A/S, Bagsvaerd, Hovedstaden DK 2880, Denmark.
Many biological disciplines rely upon the transformation of host cells with heterologous DNA to edit, engineer, or examine biological phenotypes. Transformation of model cell strains () under model conditions (electroporation of circular supercoiled plasmid DNA; typically pUC19) can achieve >10 transformants/μg DNA. Yet outside of these conditions, e.
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