DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.
Abstract Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur–DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur– feoAB1 operator complex and the Fur– Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.
This paper studies the formation and succession of the marine microfou-ling organisms community.The glass and stainless sheets were exposed to natural sea water in the Xiamen Harbor for nineteen days.The fouling organism which first appeared in an hour's exposure was bacteria while in the course of the exposure the dominant species were bacteria and diatoms.Then,after one day's exposure there appeared some diatoms (such as Coco-neis scutellum Ehr.,Licomphora flabellata Gram.) and fungi.As time went on,bacteria,diatoms,filamentous algae and other fouling organisms became more apparent whereas there was no marked increase of the fungi.What accompanied this process after a week's exposure was a loss of surface luster of the exposed sheets and the formation of a visible thin fouling layer,which then developed into notable gross surface and apparent layering.The second layer of microorganisms which were attached to the sheets consisted mainly of non-motile diatoms and filamentous.Bacteria isolated from the surface of the glass and stainless steel sheets were seven genera,that is Pseudomona,Staphylococcus,Bacillus,Xanthomonas,Flavobacterium,Micrococcus and Corynebacterium.About twenty-one genera of dia-toms(sixty-one species)were identified from the sheets that had been exposed for nineteen days.Besides there were also fungi such as Aspergillus,Penici-llium,Trichoderma,Cladosporium and yeasts such as Sterigmatomyces,Crytococcus Rhodotorula.Actinomyces isolated were only Streptomyces which were few in number.
Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (
The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine and for uncovering the genomic contributions to diseases. The Sanger sequencing method, although enormously productive for more than 30 years, requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs. The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform. Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3′-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3′-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface via click chemistry is unambiguously identified with this chip−SBS system. The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3′-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrifice of a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3′-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3′-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3′-capped NRTs. It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care.
An autoantibody specific for a DNA quadruplex structure has been isolated and cloned from three-month-old autoimmune "viable motheaten" mice. This antibody (mev-alpha Q1) has been tested extensively in vitro and found to bind specifically and preferentially to the parallel-stranded quadruplex structure formed by the oligonucleotide d(CGC G4 GCG). The anti-quadruplex antibody does not show specific affinity for single-, double-, or triple-stranded oligonuclotides of similar CG-rich sequence motifs.
Summary Precise insertion of DNA sequences into specific genome locations is essential for genome editing. Current Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)–CRISPR associated protein (Cas) protocols rely on homology-directed repair (HDR). These protocols require laborious vector construction and suffer from low efficiency. Oligo DNA can be used as donor DNA (DD) for precise DNA insertion, or targeted insertion (TI) via nonhomologous end joining (NHEJ) in many species. Here, we report a simple protocol that eliminates the need for expensive equipment and vector construction by using polyethylene glycol (PEG) to deliver non-modified synthetic single-stranded oligo DNA (ssODN) and CRISPR-Cas9 ribonucleoprotein (RNP) into protoplasts. Up to 50.0% targeted insertion was achieved in Nicotiana benthamiana and 13.6% in Rapid Cycling Brassica oleracea (RCBO) without antibiotic selection. Using 60 nt DD that contained 27 nt homologous arms, 6 out of 22 regenerated plants showed TI, and one of them had a precise insertion of 6 bp EcoRI (4.5%) in N. benthamiana. Based on whole-genome sequencing, DD inserted only in the double-strain break (DSB) positions that were induced by the CRISPR-Cas RNP. Importantly, the analysis of T1 progenies indicated that the TI sequences were successfully transmitted into the next generation.
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