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。     

借助纳米珠制作单分子DNA芯片

传统的单分子DNA芯片制作技术主要依靠稀溶液铺设,有很大的随机性,即有许多DNA分子重叠而无法观察,还有一部分基片上没有样品,造成浪费,为克服上述缺点,作者采用了一种新的单分子芯片铺设技术,纳米珠作为单分子DNA的连接载体,利用其空间位阻作用将DNA分子沉降到经过表面化学处理的基片表面,纳米珠的直径和DNA长度控制芯片上样点间距,去除纳米珠后,获得单分子DNA纳米阵列芯片。为单分子、高通量DNA芯片的制作做了新的探索。这个技术的完善将为今后单分子DNA研究开辟新的途径。     

生物素化DNA与链霉亲和素绑定初步研究

将生物素标记的DNA与链霉亲和素(streptavidin,STV)按4∶1摩尔比连接,用原子力显微镜(atomic force microscope,AFM)对混合物表征,观察到所有可能的几种DNA-STV复合物。并按照不同摩尔浓度比和孵育时间配制DNA与STV混合液,探讨了影响结合效率的因素,初步得到用生物素-链霉亲和素系统批量制备1STV-1DNA单分子复合物的最佳制备条件,为未来单分子DNA的大规模快速制备奠定了基础。     

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。     

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.     

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，     

硫代乙酸/DNA修饰金电极的电化学行为研究

采用循环伏安法,在K3Fe(CN)6/K4Fe(CN)6溶液体系中,分别对裸金电极,硫代乙酸自组装单分子膜(Self-assembled monolayers:SAMs)修饰的金电极、二次自组装技术制备的Au/硫醇/DNA混合双层膜(Hybrid Bilayer Membranes,HBMs)修饰的金电极的电化学行为进行了表征。结果表明,硫代乙酸自组装单分子膜使DNA和可能与DNA相互作用的生物物质避免了与金电极表面的直接接触,从而防止可能发生的变性。利用自组装单分子膜对电极的封闭作用,可以消除由吸附产生的对金属电极的毒化。     

分子印迹技术在茶叶中的应用

分子印迹技术是一项制备与识别"分子钥匙"的人工"锁"技术,在分离提纯、免疫分析、模拟酶、催化以及生物传感器等方面的应用研究有较广阔的空间。本文对分子印迹技术在提取纯化茶叶中咖啡因、儿茶素、EGCG单体等有效成分中的应用现状进行了综述,并分析了应用前景,以期为分子印迹技术与茶叶深加工技术的进一步"合作"开拓思路。     

SSR标记技术在玉米品种纯度鉴定上的利用研究

采用玉米单粒种子DNA的提取新方法,对提取的DNA经4.5%PA胶进行电泳检测。结果表明:此法具有快速、DNA分子量大、不降解和纯度高等优点,完全可以满足利用SSR分子标记进行DNA指纹分析的需要,为DNA指纹鉴定技术在玉米种子纯度及玉米分子标记辅助育种中的广泛应用提供了基础。     

The complex folding network of single calmodulin molecules

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.     

全树木片的气流分选

本文简要论述瑞典Bacho工业公司研制的新型JGB重力气流分选机的工作原理,以及对该机进行生产性试验检测的结果。 该机能把全树木片混合物分选成工业木片和燃料木片两部分,能有效地把树皮、细小碎粒和砂质分离出来。但对枝丫木片的分选能力欠佳。同时,在分选期间有部分木材损失。     

Conductance-controlled point functionalization of single-walled carbon nanotubes

We used covalent attachments to single-walled carbon nanotubes (SWNTs) to fabricate single-molecule electronic devices. The technique does not rely on submicrometer lithography or precision mechanical manipulation, but instead uses circuit conductance to monitor and control covalent attachment to an electrically connected SWNT. Discrete changes in the circuit conductance revealed chemical processes happening in real time and allowed the SWNT sidewalls to be deterministically broken, reformed, and conjugated to target species. By controlling the chemistry through electronically controlled electrochemical potentials, we were able to achieve single chemical attachments. We routinely functionalized pristine, defect-free SWNTs at one, two, or more sites and demonstrated three-terminal devices in which a single attachment controls the electronic response.     

Discrimination of bark from wood chips through texture analysis by image processing

Utilization of wood chips for bioenergy requires classification and segregation of the constituents of the chipped mass to help optimize energy conversion. Wood chips obtained from processes such as forest thinning can contain a considerable amount of material other than wood chips, such as bark. An image processing algorithm was developed to discriminate bark from wood chips. The algorithm involved object identification, image capture, single value decomposition to describe wood texture evident in grayscale image with a single numerical value, and application of logistic models involving the single values representative of wood texture to predict whether a chip is bark. The percentage of correct predictions using this system was about 98%.     

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.     

SNPS在大豆等作物遗传及改良中的应用

单核苷酸多态性(singlenucleotidepolymorphism,SNP)是指DNA序列上的单个碱基变异,它具有分布广、多态信息量大、易于检测和统计分析等优点,被称为继RFLP和微卫星标记后的第三代基因遗传标记。单核苷酸多态性是等位基因间序列差异最为普遍的类型,可作为一种高通量的遗传标记。已建立PCR扩增目标序列及其产物测序和电子SNP(eSNP)等多种发现和检测SNP的方法。大豆等作物也已开展了SNP分析。一些栽培作物种质的多样件不断减少,其结果连锁不平衡(linkagedise鄄quilibrium,LD)增加,这有利于目的基因座上SNP单元型(haplotype)与表型的相关性分析。SNP已在作物基因作图及其整合、分子标记辅助育种和功能基因组学等领域展示了广泛的应用价值。     

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.