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Highlights • Homeologous recombination in a mammalian genome is enabled by nearby homology. • The mechanism of recombination is impacted by proximity to a double-strand break. • Mismatch repair may at times facilitate homeologous recombination in mammals. Abstract We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the "recipient") rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a "donor" tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of "homeology" relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans is an important microorganism used in biomining operations for metal recovery. Whole-genomic diversity analysis based on the oligonucleotide microarray was used to analyze the gene content of 12 strains of A. ferrooxidans purified from various mining areas in China. Among the 3100 open reading frames (ORFs) on the slides, 1235 ORFs were absent in at least 1 strain of bacteria and 1385 ORFs were conserved in all strains. The hybridization results showed that these strains were highly diverse from a genomic perspective. The hybridization results of 4 major functional gene categories, namely electron transport, carbon metabolism, extracellular polysaccharides, and detoxification, were analyzed. Based on the hybridization signals obtained, a phylogenetic tree was built to analyze the evolution of the 12 tested strains, which indicated that the geographic distribution was the main factor influencing the strain diversity of these strains. Based on the hybridization signals of genes associated with bioleaching, another phylogenetic tree showed an evolutionary relationship from which the co-relation between the clustering of specific genes and geochemistry could be observed. The results revealed that the main factor was geochemistry, among which the following 6 factors were the most important: pH, Mg, Cu, S, Fe, and Al. [ABSTRACT FROM AUTHOR]

In this paper, we demonstrate a fluorescence immunoglobulin E (IgE) assay probe based on a DNA aptamer. A Texas red-labeled short DNA strand (T-DNA) complementary with part of the IgE aptamer sequence was used to produce the fluorescence enhancement effected upon the binding of IgE to the aptamer. Another short DNA strand labeled with dabcyl quencher (Q-DNA) complementary with part of the aptamer sequence nearby the T-DNA location was used to lower the background fluorescence. The IgE can be detected in the concentration range from 9.2 × 10−11to 3.7 × 10−8mol L−1with a detection limit of 5.7 × 10−11mol L−1. [ABSTRACT FROM AUTHOR]

An electrochemical DNAzyme sensor for sensitive and selective detection of lead ion (Pb2+) has been developed, taking advantage of catalytic reactions of a DNAzyme upon its binding to Pb2+ and the use of DNA-Au bio-bar codes to achieve signal enhancement. A specific DNAzyme for Pb2+ is immobilized onto an Au electrode surface via a thiol-Au interaction. The DNAzyme hybridizes to a specially designed complementary substrate strand that has an overhang, which in turn hybridizes to the DNA-Au biobar code (short oligonucleotides attached to 13 nm gold nanoparticles). A redox mediator, Ru(NH3)63+, which can bind to the anionic phosphate of DNA through electrostatic interactions, serves as the electrochemical signal transducer. Upon binding of Pb2+ to the DNAzyme, the DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal of the substrate strand along with the DNA bio-bar code and the bound Ru(NH3)63+ from the Au electrode surface. The release of Ru(NH3)63+ results in lower electrochemical signal of Ru(NH3)63+ confined on the electrode surface. Differential pulse voltammetry (DPV) signals of Ru(NH3)63+ provides quantitative measures of the concentrations of Pb2+, with a linear calibration ranging from 5 nM to 0.1 μM. Because each nanoparticle carries a large number of DNA strands that bind to the signal transducer molecule Ru(NH3)63+, the use of DNA-Au bio-bar codes enhances the detection sensitivity by five times, enabling the detection of Pb2+ at a very low level (1 nM). The DPV signal response of the DNAzyme sensor is negligible for other divalent metal ions, indicating that the sensor is highly selective for Pb2+. Although this DNAzyme sensor is demonstrated for the detection of Pb2+, it has the potential to serve as a general platform for design sensors for other small molecules and heavy metal ions. [ABSTRACT FROM AUTHOR]

Guanine-rich DNA sequences commonly form helical quadruplex structures via Hoogsteen hydrogen bonds. The aggregation behavior of the nanoparticles, which are functionalized with four-guanine-terminated 27-base sequences at a nanoparticle-to-DNA ratio of 1:60, is investigated. To some extent, the guanine-quadruplex structures between the gold nanoparticles (GNPs) promote nanoparticle aggregation. However, the coordination site of the metal ion on the nanoparticle surface is partially passivated: the stability of guanine-rich DNA-GNPs is slightly lower than that of the usual DNA-GNPs, and the metal-ion specificity of nanoparticle assembly is substantially decreased. Thus, a mechanism for the aggregation of guanine-rich sequence-modified GNPs is proposed. It is possible to obtain a stable guanine-rich sequence-functionalized nanoparticle solution at high ionic strength by regulating guanine-rich DNA sequences. The controllability of guanine-rich sequence-modified nanoparticles makes the secondary structure of DNA a potentially useful candidate for DNA analysis and disease diagnostics. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nueclic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptamenc system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a selt-blocked tluoresceflt bifunctional oligonudeotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 x 10-10 M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme ass proof of concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis. [ABSTRACT FROM AUTHOR]

It is well-known that Zr4+ could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (-PO32--Zr4+-PO32--). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr4+ via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr4+ to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a "turn-on" excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr4+. This allows detection of Zr4+ with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr4+ in river water samples with satisfactory result. [ABSTRACT FROM AUTHOR]

This work reports the development of a new molecular beacon-based junction sensing system with highly Sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNAcleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system. [ABSTRACT FROM AUTHOR]

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical. [ABSTRACT FROM AUTHOR]

A novel electrochemical sensor was developed for sensitive and selective detection of mercury(II), based on thymine-Hg2+-thymine (T-Hg2+-T) coordination diemistiy. This strategy exploited the cooperativity of proximate poly-T oligonucleotides in coordination with Hg2+. Ferrocene (Fc)-tagged poly-T oligonucleotides were immobilized on the electrode surface via self-assembly of the terminal thiol moiety. In the presence of Hg2+, a pair of poly-T oligonucleotides could cooperatively coordinate with Hg2+, which triggered a conformational reorganization of the poly-T oligonucleotides from flexible single strands to relatively rigid duplex like complexes, thus drawing the Fe tags away from the electrode with a substantially decreased redox current. The response characteristics of the sensor were thoroughly investigated using capillary electrophoresis and electrochemical measurements. The results revealed that the sensor showed a sensitive response to Hg2+ in a concentration range from 1.0 nM to 2.0 μM, with a detection limit of 0.5 nM. Also, this strategy afforded exquisite selectivity for Hg2+ against a reservoir of other environmentally related metal ions, compared to existing anodic stripping voltammetry (ASV) techniques. In addition, this sensor could be implemented using minimal reagents and working steps with excellent reusability through mild regeneration procedure. It was expected that this cost-effective electrochemical sensor might hold considerable potential in on-site applications of Hg2+ detection. [ABSTRACT FROM AUTHOR]