Single nucleotide variation detection on 3D DNA microarray by ligation of two-terminal-modified universal probes

Exploiting the advantages of three-dimensional (3D) DNA microarray, we put forward a novel strategy termed "ligation of two-terminal-modified universal probes" for single nucleotide variation (SNV) detection on 3D microarray. By performing specific ligation reaction between the unmodified hybridization primer with 3' hydroxyl terminus and the universal probe with phosphorylated 5' terminus and fluorescently labeled 3' terminus, two point mutations (C3206T and A5301G) in the PCR products immobilized on the 3D polyacrylamide gel DNA microarray were accurately discriminated. This method can not only maintain the predominance of 3D DNA microarray as a platform with high through-put, but also can exert this predominance to the cases of detecting a small quantity of samples and multiple SNVs since four universal probes can be employed to detect all SNVs, therefore, it is more feasible for laboratory research and provide an effective tool for clinical diagnosis.     

Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore

Nanopores have been used as extremely sensitive resistive pulse sensors to detect analytes at the molecular level. There has been interest in using such a scheme to rapidly and inexpensively sequence single molecules of DNA. To establish reference current levels for adenine, cytosine, and thymine nucleotides, we measured the blockage currents following immobilization of single-stranded DNA polyadenine, polycytosine, and polythymine within a protein nanopore in chemical orientations in which either the 3' or the 5' end enters the pore. Immobilization resulted in low-noise measurements, yielding sharply defined current distributions for each base that enabled clear discrimination of the nucleotides in both orientations. In addition, we find that not only is the blockage current for each polyhomonucleotide orientation dependent, but also the changes in orientation affect the blockage currents for each base differently. This dependence can affect the ability to resolve polyadenine and polythymine; with the 5' end entering the pore, the separation between polyadenine and polythymine is double that observed for the 3' orientation. This suggests that, for better resolution, DNA should be threaded through the 5' end first in nanopore DNA sequencing experiments.     

Conformational switching immobilized hairpin DNA probes following subsequent expanding of gold nanoparticles enables visual detecting sequence-specific DNA

A simple, rapid, and sensitive method for visual detection of sequence-specific DNA was developed using hairpin DNA as the recognition element and hydroxylamine-enlarged gold nanoparticles (Au-NPs) as the signal producing component. In the assay, we employed a hairpin DNA probe dually labeled with amine and biotin at the 5'- and 3'-end, respectively. The probe was coupled with reactive N-oxysuccinnimide in a DNA-bind 96-well plate. Without the target DNA, the immobilized hairpin probe was in a "closed" state, which kept the streptavidin-gold off the biotin. The hybridization between the loop sequence and the target broke the short stem duplex upon approaching the target DNA. Consequently, biotin was forced away from the 96-well plate surface and available for conjugation with the streptavidin-gold. The hybridization could be detected visually after the HAuCl(4)-NH(2)OH redox reaction catalyzed by the Au-NPs. Under the optimized conditions, the visual DNA sensor could detect as low as 100 amol of DNA targets with excellent differentiation ability and even a single-base mismatch.     

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).     

Pentafluorinated probes for the X-ray photoelectron spectroscopic study of immobilized bifunctional silanes

Bifunctional silanes constitute valuable linking agents for attachment of biomolecules at high levels of surface population density through the formation of thioether or disulfide bonds. Three such compounds, 1-((trifluoroacetyl)-thio)-11-(trichlorosilyl)undecane, 1-bromo-11-(trichlorosilyl)undecane, and 1-((bromoacetyl)oxy)-11-(trichlorosilyl)undecane, are discussed in terms of their surface chemistry on silicon wafers. To examine the electrophilic and nucleophilic generation of sulfur-containing linkages, three new probes, N'-(pentafluorophenyl)iodoacetohydrazide, N'-(pentafluorophenyl)-3-(2-pyridylthio)propriono-hydrazide, and N'-(pentafluorophenyl)mercaptoacetohydrazide, are introduced with respect to their reactions with silanized surfaces (studied by X-ray photoelectron spectroscopy). Thiol-functionalized surfaces obtained by silanization act as nucleophiles toward the probes. In air, low yields of conjugation are exhibited which are attributed to the unavailability of thiol groups because of intramolecular disulfide group formation instigated by oxygen or by disulfide exchange with the proprionyl probe. The behavior of electrophilic silanized surfaces toward the mercaptoacetyl-containing probe is governed by the nature of the leaving group and by steric factors.     

The label-free unambiguous detection and symbolic display of single nucleotide polymorphisms on DNA origami

Single nucleotide polymorphisms (SNPs) are the most common genetic variation in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair inhibits the progress of branch migration in the direction of the mispair. We have combined the effectiveness of kinetic methods with atomic force microscopy of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four nucleotide alphabetic characters, A, T, G and C, and the symbol containing the test nucleotide identity vanishes in the presence of the probe. The system also works with pairs of probes, corresponding to heterozygous diploid genomes.     

Microscope objective for large-angle fluorescence used for rapid detection of single nucleotide polymorphisms in DNA hybridization

A new type of microscope objective is used for the rapid detection of sequence-dependent affinity variations in DNA hybridization. We demonstrate that by performing probe/target hybridization on coverslips at room temperature terminal SNPs (single nucleotide polymorphisms) can be detected within seconds. The study of weak pair interaction, such as the association of very short DNA oligomers, requires the use of high analyte concentrations of both partners to generate a detectable amount of associated pairs. The background of high concentrations of unbound fluorescing analyte can easily hide the low signal of a weakly affine reaction and makes association extremely difficult to detect. Fluorescence detection is a powerful approach to analyze minute amounts of material, even single molecules, but it is usually limited to rather low concentrations. This limitation is now overcome due to the new type of microscope objective, which produces an extremely small detection volume at a water/glass interface.     

Comparative characterization to the three kinds of solid-shape FeS

The microstructures of three kinds of the synthetical solid FeS, acting as the solid lubricant, which includes FeS bulk, FeS particles and FeS powder, were studied in this article. Their surface morphologies were investigated with SEM, and XRD was used to study the phase structures, TEM was utilized to observe the micro-morphologies and analyze the microstructures. The results showed that the crystals of solid FeS were composed of many hexagonal single crystals and multi-crystals, with two kinds of crystalline structure, hexagonal structure and face-centered cubic structure. The surface of FeS was in the shape of a floccule except for a little granular figure. The micro-morphology of FeS presented irregular flakes which can be seen from TEM pictures, the size of grains ranging between 0.1 μm and 3 μm.     

Boron-doped diamond single crystals for probes of the high-vacuum tunneling microscopy

The results of studying the structure of diamond single crystals grown by the temperature gradient method with the aim to obtain samples having maximum uniform characteristics for manufacturing probes for scanning electron microscopes with a specified axial orientation and controlled distribution of the dopant have been considered. It has been shown that the use of similar probes in scanning tunneling microscopy decreases the probability of incidental tunneling channels with participation of the surface states caused by the presence of boron atoms in the diamond structure and increases the reliability of experimental data. The high stability of monocrystalline diamond probes and the possibility to attain the atomic resolution with the help of them have been demonstrated by the investigations of the (0001) graphite plane using scanning tunneling microscopy.     

Structure-function relationships in high-density octadecylsilane stationary phases by Raman spectroscopy. 3. Effects of self-associating solvents

Raman spectroscopy is used to examine the subtle effects of polar, hydrogen-bonding solvents; temperature; and the surface grafting method (surface- or solution-polymerized) on alkyl chain rotational and conformational order in a series of high-density octadecylsilane stationary phases ranging in surface coverage from 3.09 to 6.45 micromol/m2. Rotational and conformational order is assessed using the intensity ratio of the antisymmetric to symmetric v(CH2) modes as well as the frequencies at which these Raman bands are observed. Alkyl rotational and conformational order decreases with decreasing surface coverage in these polar solvents, consistent with the behavior of these materials in air. For homogeneously distributed, high surface coverage materials, these polar solvents induce rotational ordering that is proposed to be due to the self-association of these solvents through hydrogen bonding or other dipole interactions at the alkylsilane-solvent interface. From these observations, molecular pictures of these solvent-stationary-phase interfaces are proposed in which solvent interaction with the stationary phase occurs primarily at the distal methyl group of the alkyl chains.     

三种绝缘子材料的分析及其研究进展

论述了绝缘子对于电力系统稳定运行的重要意义.详细介绍了陶瓷绝缘子、玻璃绝缘子以及复合绝缘子所用材料的化学组成与结构.根据其化学组成的不同对各绝缘子进行了分类介绍,并分析了化学组成对于绝缘子电气性能与机械性能的影响.介绍了外部作用对于绝缘子性能的影响,尤其详细描述了电晕、紫外线等外力作用对硅橡胶绝缘子化学结构的影响及其机理.     

Structure-function relationships in high-density octadecylsilane stationary phases by Raman spectroscopy. 2. Effect of common mobile-phase solvents

Raman spectroscopy is used to examine the effects of solvent, temperature, and surface grafting method (surface or solution polymerized) on alkyl chain rotational and conformational order in a series of high-density octadecylsilane stationary phases ranging in surface coverage from 3.09 to 6.45 micromol/m2. Rotational and conformational order is assessed using the intensity ratio of the antisymmetric to symmetric v(CH2) modes as well as the frequency at which these Raman bands are observed. Solvents studied include perdeuterated hexane, toluene, chloroform, tetrahydrofuran, benzene, methanol, acetone, acetonitrile, and water. Stationary-phase order was investigated at temperatures between 258 and 323 K. Alkyl chain rotational and conformational order, and hence, solvation of the stationary phase, is dependent on solvent parameters (polarity, size, etc.), temperature, and stationary-phase properties (polymerization method and surface coverage). Information on stationary-phase conformational order allows solvent-stationary-phase interactions to be described in terms of a combination of adsorption and partitioning models for reversed-phase liquid chromatography. Finally, a distinct interplay between solvent- and temperature-induced ordering of these stationary phases is documented that is also a function of solvent and stationary-phase properties.     

Weibull analysis of strength-length relationships in single Nicalon SiC fibres

Nicalon SiC fibre is naturally brittle and offers high-temperature application in fibrous composites. Due to the randomly distributed flaws along the fibre, the statistical variability in single-fibre strength is obvious. In this paper, the effect of heat-cleaning procedures on Nicalon fibres has been investigated, and the statistical strength and variability of single Nicalon fibres have been characterized in tension and compared. Experimental results show that the strengths of single Nicalon fibres among the three types of heat-cleaning procedures are less than that of as-received unsized fibres by 22–30%. In addition, both the failure load and the failure stress of the fibres, for a given gauge length (50 mm) and yarn cross-section, can be well fitted to a two-parameter Weibull distribution. The effect of gauge length over the range from 10–175 mm, holding the strain rate constant, was also studied. The logarithmic strength-length plots show that the strength of single Nicalon fibres follows the weakest-link rule.     

三种翅片管在空冷器应用中的性能比较与分析

通过实验，对缠绕式翅片管、外翅片铸铁管和外翅片铸铝管在低温空冷器应用中的传热性能进行比较和分析。实验表明，由普通缠绕式翅片管制成的空冷器比铸管制成的空冷器在短时期内的传热系数大，并且对管内流体的降温效果非常明显；铸铝管空冷器的散热能力并不比铸铁管空冷器强很多。     

3种苯基脲类除草剂的THz光谱特性研究

利用太赫兹时域光谱(THz-TDS)技术对3种苯基脲类除草剂,敌草隆、伏草隆、绿麦隆的标准品分别进行光学参数的测试,获得样品在有效THz波段范围的折射率谱和吸收谱.实验结果发现,三种样品的吸收谱均有明显的特征吸收峰,且它们之间存在很大差异,平均折射率分别为1.593、1.474、1.504.利用Gaussian03的B3LYP函数在6-311G(d)基组水平上计算其单分子的振动频率,模拟结果与实验数据存在一定差异,说明单分子模拟存在局限性.利用DMol3程序的BP函数,在DNP基组水平上进行晶体振动频率模拟,模拟结果与实验数据一致性较好.通过实验和理论分析,THz-TDS技术可作为鉴别这三种除草剂的一种有效手段.     

Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes

Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.     

Study of single-stranded DNA binding protein-nucleic acids interactions using unmodified gold nanoparticles and its application for detection of single nucleotide polymorphisms

We have developed a label-free homogeneous phase bioassay to characterize the DNA binding properties of single-stranded DNA binding (SSB) protein, a key protein involved in various DNA processes such as DNA replication and repair. This assay uses gold nanoparticles (AuNPs) as sensing probe and is based on the phenomenon that preformed SSB-single-stranded DNA (ssDNA) complexes can protect AuNPs against salt-induced aggregation better than SSB or ssDNA alone. With the controlled particle aggregation/dispersion as measure, this assay can be used to detect the formation of SSB complexes with ssDNA of different length and nucleotide composition and to assess their binding properties without tedious and complicated assay procedures. On the basis of the inverse relationship between DNA hybridization efficiency and the tendency of SSB to form protection complex with unhybridized ssDNA to AuNPs, this assay is further developed to detect DNA hybridization with single nucleotide polymorphism selectivity. Owing to the high affinity between SSB and dissociated ssDNA, single-base mismatch discrimination in a long sequence of 30-mer DNA was achieved for both the end- and center-base mismatch. Unlike the conventional techniques for DNA and protein analysis, current AuNPs-based sensing strategy is simple in design, fast in detection, and economical for operation without the need of sophisticated equipment.     

Structure-function relationships in high-density octadecylsilane stationary phases by Raman spectroscopy. 4. Effects of neutral and basic aromatic compounds

The effects of aromatic compounds (toluene, benzene, p-xylene, anisole, aniline, and pyridine), temperature, and surface grafting method (surface- or solution-polymerized) on alkyl chain rotational and conformational order in a series of high-density octadecylsilane stationary phases ranging in surface coverage from 3.09 to 6.45 micromol/m2 are examined by Raman spectroscopy. Rotational and conformational order are assessed using the intensity ratio of the antisymmetric to symmetric v(CH2) modes as well as the frequency at which the symmetric v(CH2) band is observed. Alkyl rotational and conformational order decrease with decreasing surface coverage in these aromatic compounds, which is consistent with the behavior of these materials in air and in other solvents. In addition, order of the alkyl chains is dependent on solvent hydrophobicity, hydrogen-bonding ability, and basicity. The most hydrophobic compounds impart disorder to the stationary phase; the hydrogen-bonding aromatics increase the rotational order of homogeneously distributed, high-surface-coverage materials; and basic aromatic compounds increase the conformational order of high- and low-coverage materials as the basic compounds undergo silanophilic interactions with exposed surface silanols. From these observations, molecular pictures of the chromatographic interface that display interactions between the alkyl chains and these aromatic compounds are proposed.     

Implications of the dependence of the elastic properties of DNA on nucleotide sequence

Recent advances in structural biochemistry have provided evidence that not only the geometric properties but also the elastic moduli of duplex DNA are strongly dependent on nucleotide sequence in a way that is not accounted for by classical rod models of the Kirchhoff type. A theory of sequence-dependent DNA elasticity is employed here to calculate the dependence of the equilibrium configurations of circular DNA on the binding of ligands that can induce changes in intrinsic twist at a single base-pair step. Calculations are presented of the influence on configurations of the assumed values and distribution along the DNA of intrinsic roll and twist and a modulus coupling roll to twist. Among the results obtained are the following. For minicircles formed from intrinsically straight DNA, the distribution of roll-twist coupling strongly affects the dependence of the total elastic energy Psi on the amount alpha of imposed untwisting, and that dependence can be far from quadratic. (In fact, for a periodic distribution of roll-twist coupling with a period equal to the intrinsic helical repeat length, Psi can be essentially independent of alpha for -90 degrees < alpha <90 degrees.) When the minicircle is homogeneous and without roll-twist coupling, but with uniform positive intrinsic roll, the point at which Psi attains its minimum value shifts towards negative values of alpha. It is remarked that there are cases in which one can relate graphs of Psi versus alpha to the 'effective values' of bending and twisting moduli and helical repeat length obtained from measurements of equilibrium distributions of topoisomers and probabilities of ring closure. For a minicircle formed from DNA that has an 'S' shape when stress-free, the graphs of Psi versus alpha have maxima at alpha = 0. As the binding of a twisting agent to such a minicircle results in a net decrease in Psi, the affinity of the twisting agent for binding to the minicircle is greater than its affinity for binding to unconstrained DNA with the same sequence.     

Selection of a reference sequence for the determination of nucleotide sequences in DNA molecules

A fragment of the DNA sequence of the pUC18 plasmid is proposed as a reference sequence for the calibration and metrological assurance of genome analyzers. This fragment consists of 271 nucleotide pairs and is obtained by a reference method. This sequence is optimal for determining the metrological characteristics of genome analyzers in light of its stability and the existence of several segments with repeating nucleotides. An example is given for the calculation of a genome analyzer characteristic, namely, the probability of error in measurement of the genome sequence.