单粒微米珠-单根DNA复合物的微流控分离初步研究

定点单分子DNA芯片有重要用途,但其制备极具挑战性,用光镊、磁镊及原子力显微镜等方法分离单分子DNA,为此建立的平台设备昂贵,且分离效率太低,难以满足实验要求。本研究采用微流控技术,在层流基础上电泳分离与富集单粒微米珠-单根DNA的复合物,旨在为基于微米孔阵列承载这种复合物的定点单分子DNA芯片的制备提供样本。此技术的完善将为单分子DNA芯片制作开辟新的途径。     

单分子DNA在亲水性基底表面的拉伸研究

报道了一种在亲水性表面拉伸裸露λDNA和DNA-微球复合物中λDNA的简单方法。将样品滴在水虎鱼溶液(piranha)和碱性过氧化氢溶液(RCA)清洗的盖玻片上,通过液滴展布与蒸发,利用静电力和表面张力相互作用,将DNA分子伸展并平铺在玻片表面上。激光共聚焦荧光显微镜成像表明,该方法可以较好地将这两种状态的DNA拉直,拉伸长度大于氨基硅烷修饰的表面拉伸方法,同时,也避免了以往一些分子梳过度拉伸而造成DNA变性的弊端。该方法为在单分子水平研究DNA的物理特性和DNA-蛋白质的相互作用提供了新的途径,也为大规模操纵单分子DNA提供了可能。     

二氧化硅生物荧光纳米颗粒的制备与应用

以荧光染料(联吡啶钌配合物)为核,二氧化硅为外壳制备了荧光纳米颗粒.电镜检测结果显示,合成的二氧化硅纳米颗粒粒径为(20±3)nm,分布均匀,形态规则.对荧光纳米颗粒进行生物修饰得到荧光探针,以DNA碱基配对原则为基础建立检测DNA的方法,利用激光扫描共聚焦显微镜研究玻片上的荧光信号和荧光强度.结果表明,该方法不仅可定量检测到4.4×10-8 mol/L的目标DNA3,而且可定性检测目标DNA3、单碱基错配DNA4及随机序列DNA5,为发展DNA检测技术提供了新的思路.     

DNA芯片技术综述

1996年底，美国加州旧金山Affymetrix公司Fodor等从固相支持物上合成多肽中得到启发，灵活运用了照相平版印刷、计算机、半导体、激光共聚焦扫描、寡核苷酸合成、荧光标记、DNA分子杂交及分子生物学的其它技术创造了世界上第一块DNA芯片。DNA芯片的出现充分体现了生物学技术与其他学科和技术的相互交叉和渗透。DNA芯片技术是融微电子学、生命科学、物理学于一体的一项崭新技术，     

森林植物分子生态学的研究方法

森林植物分子生态学的核心内容是检测其群落、种群、个体水平上的遗传多样性．较详细地综述了DNA水平上森林植物分子生态学的研究方法;DNA分子标记、DNA测序、基因克隆技术、DNA芯片技术的的原理和方法；简要介绍了蛋白质水平上的研究方法：等位酶分析和蛋白质组学的原理和方法．     

微生物分子生态学技术及其在食品产业中的应用前景

综述了新兴的不依赖于培养的微生物分子生态学技术,包括两类：不基于微生物总基因组DNA的分析方法,主要有单碳源利用图谱法和磷脂脂肪酸图谱法;基于微生物总基因组DNA的分子分析方法,主要有克隆文库分析法、宏基因组文库技术、G＋C含量分析法、总DNA复性动力学分析法、群落水平总基因组DNA交互杂交法、荧光原位杂交技术、DNA微阵列芯片技术、变性/温度梯度凝胶电泳、单链构象多态性分析、限制片段长度多态性/扩增核糖体DNA限制性分析、末端标记限制片段长度多态性、核糖体基因间隔区分析、随机扩增多态性DNA等.并提出了这套技术在食品产业中的应用前景：发现新菌种,生产新型酶制剂,优化改造新工艺,确保食品质量与安全等,为食品产业的继续开发提供新的技术支持.     

微生物分子生态学技术及其在食品产业中的应用前景

综述了新兴的不依赖于培养的微生物分子生态学技术,包括两类：不基于微生物总基因组DNA的分析方法,主要有单碳源利用图谱法和磷脂脂肪酸图谱法;基于微生物总基因组DNA的分子分析方法,主要有克隆文库分析法、宏基因组文库技术、G＋C含量分析法、总DNA复性动力学分析法、群落水平总基因组DNA交互杂交法、荧光原位杂交技术、DNA微阵列芯片技术、变性/温度梯度凝胶电泳、单链构象多态性分析、限制片段长度多态性/扩增核糖体DNA限制性分析、末端标记限制片段长度多态性、核糖体基因间隔区分析、随机扩增多态性DNA等.并提出了这套技术在食品产业中的应用前景：发现新菌种,生产新型酶制剂,优化改造新工艺,确保食品质量与安全等,为食品产业的继续开发提供新的技术支持.     

基因芯片技术及其在烟草改良中的应用前景

讨论了DNA芯片制作的基本原理和主要步骤，介绍了基因芯片的主要类型。探讨了基因芯片技术在转基因烟草检测、烟草基因表达与调控研究、烟草种子检测和烟草分子育种中的应用前景。     

紫外光诱导硅表面寡聚乙二醇单分子膜的制备

研究报道了一种表面具有抗蛋白吸附性能硅材料的制备方法。该方法利用紫外光线（波长为254 nm）诱导硅氢烷基化反应机理,将寡聚乙二醇分子共价偶联到硅氢化表面上;采用多种表面分析手段（X射线能谱仪、原子力显微镜、椭圆偏振光谱仪、接触角测量仪等）对所得材料进行综合表征,结果显示寡聚乙二醇分子在硅表面上形成了一层致密的单分子膜。最后试验通过测试寡聚乙二醇膜对纤维蛋白原的吸附性能,表征了修饰后硅表面抗蛋白吸附和热稳定性能。结果表明,寡聚乙二醇单分子膜不仅具有良好的抗蛋白吸附性能,而且在生理环境下可以保持长期的稳定性。由此可以推断,这种表面具有抗蛋白吸附性能的硅材料将在药物载体、蛋白检测芯片、生物微机电系统以及生物传感器等研究领域具有一定的应用价值。     

用单价STV制备单分子蛋白质-DNA复合物的研究

将生物素结合位点失活的链霉亲和素(streptavidin,STV)变异体变性后的亚基与野生型STV变性后的亚基以摩尔比3∶1的比例混合,然后重折叠复性,镍柱层析分离得到只有一个有活性生物素结合位点的突变体STV,即单价STV。将单价STV与500bp一端生物素标记的双链DNA(biotin-DNA)按不同摩尔比混合孵育,探讨使用单价STV制备1STV-1DNA复合物的可行性,并筛选制备1STV-1DNA复合物的最佳混合摩尔比。结果表明,在所用的摩尔比梯度中,单价STV与biotin-DNA混合的最佳摩尔比为1∶1,与使用野生型STV制备方法相比,使用单价STV更为简便、高效,为单分子DNA操纵、单分子DNA-蛋白质相互作用研究以及单分子DNA芯片的制备等提供了便捷的方法。     

Fluorescence spectroscopy of single biomolecules

Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.     

Inducing and viewing the rotational motion of a single molecule

Tunneling electrons from the tip of a scanning tunneling microscope were used to induce and monitor the reversible rotation of single molecules of molecular oxygen among three equivalent orientations on the platinum(111) surface. Detailed studies of the rotation rates indicate a crossover from a single-electron process to a multielectron process below a threshold tunneling voltage. Values for the energy barrier to rotation and the vibrational relaxation rate of the molecule were obtained by comparing the experimental data with a theoretical model. The ability to induce the controlled motion of single molecules enhances our understanding of basic chemical processes on surfaces and may lead to useful single-molecule devices.     

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.     

Zero-mode waveguides for single-molecule analysis at high concentrations

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.     

Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization

The classical view of DNA posits that DNA must be stiff below the persistence length [<150 base pairs (bp)], but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free assay for studying the cyclization of single DNA molecules in real time. The assay samples the equilibrium population of a sharply bent, transient species that is entirely suppressed in single-molecule mechanical measurements and is biologically more relevant than the annealed species sampled in the traditional ligase-based assay. The looping rate has a weak length dependence between 67 and 106 bp that cannot be described by the worm-like chain model. Many biologically important protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA.     

Probing gene expression in live cells, one protein molecule at a time

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.     

Examining nanoenvironments in solids on the scale of a single, isolated impurity molecule

Optical spectroscopy of single impurity molecules in solids can be used as an exquisitely sensitive probe of the structure and dynamics of the specific local environment around the single molecule (the "nanoenvironment"). Recently observed effects such as spectral diffusion, perturbations by external fields, changes in molecular photophysics, shifts in vibrational modes, optical modification of the absorption spectrum, dynamics due to amorphous system physics, and magnetic resonance of a single molecular spin attest to the vitality of and growing interest in this new field, which may lead to optical storage on the single-molecule level.     

Reproducible measurement of single-molecule conductivity

A reliable method has been developed for making through-bond electrical contacts to molecules. Current-voltage curves are quantized as integer multiples of one fundamental curve, an observation used to identify single-molecule contacts. The resistance of a single octanedithiol molecule was 900 +/- 50 megohms, based on measurements on more than 1000 single molecules. In contrast, nonbonded contacts to octanethiol monolayers were at least four orders of magnitude more resistive, less reproducible, and had a different voltage dependence, demonstrating that the measurement of intrinsic molecular properties requires chemically bonded contacts.     

Polymerase exchange during Okazaki fragment synthesis observed in living cells

DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.