DRE顺式作用元件dsDNA芯片制备

DRE顺式作用元件能与DREB转录因子特异结合,在诱导逆境（干旱、高盐、低温）基因表达过程中起重要作用。dsDNA（double strand DNA）微阵列芯片技术能够有效地检测序列特异性DNA结合蛋白质（转录因子）与大量DNA靶点（顺式作用元件）的特异性结合,可有效分析生物分子结合作用。根据DRE顺式作用元件核心序列设计并化学合成含发夹结构的单链DNA探针,采用Taq DNA聚合酶在片延伸,并对其在片延伸体系的反应温度、Mg^2＋浓度以及单链探针是否变性等条件进行了优化。结果表明,50％的反应温度,2．5mmol／L的Mg^2＋浓度和单链不变性是TaqDNA聚合酶在片延伸的最佳条件。优化方法制备的dsDNA芯片将更有利于DRE顺式作用元件与DREB抗逆转录因子相互作用的研究。     

质粒DNA抗原芯片检测抗双链DNA抗体的初步研究

目的:建立以质粒DNA作为抗原的检测血清中抗双链DNA(dsDNA)抗体的芯片方法,并与酶联免疫吸附实验比较,初步探讨用芯片法检测抗dsDNA抗体的临床价值。方法:将原核表达载体质粒pcDNAⅡ用质粒DNA快速抽提试剂盒提取纯化DNA后按1∶2稀释,用点样仪点在经3-氨丙基三乙氧基硅烷(APES)修饰的玻片上,温孵后用含有1%小牛血清白蛋白和2.5%蔗糖的PBST封闭,以Cy3标记的人IgG为二抗,建立检测dsDNA抗体的芯片方法,并与德国欧蒙公司生产的抗双链DNA检测ELISA试剂盒做比较,对包括58例系统性红斑狼疮(SLE)、25例干燥综合征(SS)、10例皮肌炎(DM)和7例类风湿关节炎(RA)在内的病人和60例健康人对照进行了抗dsDNA的对比检测。结果:对阳性标本的检测,与现用常规检测方法ELISA相比,芯片检测抗dsDNA的灵敏度为91.3%,特异度为90.7%,阳性预测值为89.3%,阴性预测值为92.5%;对健康对照的检测,2种方法均为阴性,符合率为100%。结论:与ELISA相比,用质粒DNA作为抗原建立的芯片方法的灵敏度和特异度较高,为今后建立同时检测多个自身抗体的芯片奠定了基础。     

基因芯片技术和蛋白质组技术在神经科学中的应用及其研究进展

基因芯片技术和蛋白质组技术是最近发展起来的高通量技术,二者的出现使同时分析神经系统的大量基因的表达和基因产物蛋白质及其相互作用网络成为可能。它们在神经科学中的应用为了解脑功能提供了前所未有的机会。一个典型的基因芯片实验包括：芯片的准备或购买、靶DNA和探针的准备或标记、标记探针与靶DNA的杂交、芯片扫描和影象信息的数据分析。蛋白质组技术较为复杂,包括蛋白质分离、鉴定和信息分析三方面的内容。其中,分离技术多种多样。若分离技术以二维电泳为基础,则该实验通常由以下步骤组成：蛋白质样品的准备、电泳分离、染胶、分离蛋白点的切除、蛋白质的酶解(常用胰蛋白酶)、质谱分析(鉴定)和数据的信息处理。本文综述这两项技术的内容和实验步骤,然后着重叙述它们在神经科学中的应用,讨论其优缺点和面临的挑战,展望其发展前景。     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

哺乳动物转录因子DNA结合谱

基因差异表达是生物发育和对刺激作出应答的分子基础,转录因子在这种基因差异表达中发挥着重要的调控作用。因此,要弄清楚转录因子调控基因差异表达的机理,就必须鉴定出它们全部的靶基因并构建其操纵的转录调控网络。对基因组DNA的序列特异性结合是转录因子调控基因转录的关键环节,因此,要鉴定转录因子的靶基因,就必须从它们与DNA相互作用的分子水平,鉴定它们能够识别并结合的全部DNA序列,即转录因子DNA结合谱。近年来随着DNA微阵列芯片和高通量DNA测序技术的产生和快速发展,出现了建立转录因子体内及体外DNA结合谱的一系列革命性的新技术,对该领域的研究带来重大影响。这些新技术主要包括建立转录因子体内DNA结合谱的染色质免疫沉淀-芯片技术(ChIP-chip)和染色质免疫沉淀-测序技术(ChIP-Seq),以及建立转录因子体外DNA结合谱的双链DNA微阵列芯片技术(dsDNA microarray)、指数富集配体系统进化-系列分析基因表达技术(SELEX-SAGE)、结合-n-测序技术(Bind-n-Seq)、多重大规模并行SELEX技术(MMP-SELEX)、凝胶迁移实验-测序技术(EMSA-Seq)和高通量测序-荧光配体互作图谱分析技术(HiTS-FLIP)。文章将对这些新技术做一综述。     

自组装蛋白质芯片——功能蛋白质组学的新工具

传统的蛋白质芯片制备需要进行繁琐的蛋白质表达与纯化。同时,由于蛋白质活性不稳定,蛋白质芯片不宜长期保存。新一代自组装蛋白质芯片,利用无细胞表达体系和DNA固定技术,能够将蛋白质即时、原位表达并固定在芯片上,有效地解决了传统蛋白质芯片的制备和保存问题。目前自组装蛋白质芯片已初步用于大规模蛋白-蛋白质相互作用的筛选,以及鉴定免疫优势抗原等研究。该文介绍了近年自组装蛋白质芯片技术的进展和应用研究。     

光标生物素HBV-DNA探针及其临床应用

本文报告了应用生物素交联光敏补骨酯素(Bp,Biotin-Psoralen)标记HBV DNA探针的动力学研究和临床应用结果。表明化学合成Bp经366nm光照与DNA发生加成反应。能标记dsDNA,也能标记ssDNA。加热可使Bp-dsDNA变性,强碱则不能使Bp-dsDNA变性。临床DNA检测结果表明,应用Bp-HBV探针杂交,DNA检出率与HBeAg符合率达99.08％,与~(32)P-HBV符合率达98.8％,提示光标记Bp-HBV探针灵敏度接近同位素探针。     

蛋白质芯片在蛋白质组学研究中的作用

蛋白质芯片是以高度并行性、高通量、微型化和自动化为特点的蛋白质组检测技术。本文综述了蛋白质芯片在蛋白质组学研究中的多种作用,包括普通蛋白质芯片在微量蛋白质分离、蛋白质与蛋白质之间以及蛋白质与其他小分子间相互作用和蛋白质定量检测方面的作用,普通蛋白质芯片通过与质谱技术、生物传感器技术的结合而拓展其应用范围,以及蛋白质组芯片、活性的蛋白质芯片在蛋白质组学研究中应用的进展。     

无细胞系统原位制备蛋白质芯片及其应用

主要介绍了目前在生物芯片表面进行蛋白质无细胞表达与定向制备蛋白质芯片的研究进展,包括各种基因植入芯片的方法、蛋白质体外不同表达的途径、蛋白质固定的策略以及可能的应用发展前景等．蛋白质芯片以其高通量、高灵敏和检测迅速等优点正成为蛋白质组学研究中的重要工具之一．蛋白质的高效表达与纯化、蛋白质在芯片表面的有效固定与蛋白质活性的保持等内容是蛋白质芯片技术发展的关键．采用纳米生物技术与无细胞表达系统,已经可以在生物芯片表面通过植入基因的方式制备相关的蛋白质芯片,从而为蛋白质芯片的原位制备开辟了新的方向．     

无细胞系统原位制备蛋白质芯片片及其应用

主要介绍了目前在生物芯片表面进行蛋白质无细胞表达与定向制备蛋白质芯片的研究进展,包括各种基因植入芯片的方法、蛋白质体外不同表达的途径、蛋白质固定的策略以及可能的应用发展前景等.蛋白质芯片以其高通量、高灵敏和检测迅速等优点正成为蛋白质组学研究中的重要工具之一.蛋白质的高效表达与纯化、蛋白质在芯片表面的有效固定与蛋白质活性的保持等内容是蛋白质芯片技术发展的关键.采用纳米生物技术与无细胞表达系统,已经可以在生物芯片表面通过植入基因的方式制备相关的蛋白质芯片,从而为蛋白质芯片的原位制备开辟了新的方向.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Identification, cloning and expression of p25, an AT-rich DNA-binding protein from the extreme thermophile, Thermus aquaticus YT-1

Although the G+C content of Thermus aquaticus YT-1 chromosomal DNA is 67.4%, regions with lower G+C content have also been observed. AT-rich DNA-binding proteins may contribute to the thermostability and biological functions of these DNA regions at Thermus growth temperatures. Using double-stranded DNA (dsDNA)-cellulose chromatography, a T.aquaticus YT-1 protein, designated as p25, was identified to bind preferentially to AT-rich DNA. The gene encoding p25 was cloned and sequenced after immunoscreening T.aquaticus YT-1 expression libraries. The deduced primary structure of p25 is 211 amino acids in length with a molecular weight of 23 225 Da. Native p25 was purified and characterized as a homodimer with modification possibly at lysine and arginine residues. Its preferential and temperature-dependent binding to AT-rich DNA was confirmed with mobility-shift DNA-binding assays. The protein was demonstrated to bind preferentially to dsDNA instead of single-stranded DNA. The binding of p25 to dsDNA also improved the thermotolerence of this protein. Overexpression study of fusion p25 suggested that the N-terminus of the protein might form the DNA-binding domain or be closely involved in DNA-binding activity.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

Interactions of the DNA polymerase X of African swine fever virus with double-stranded DNA. Functional structure of the complex

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n=10(+/-2) base-pairs, which is significantly shorter than the total site-size of 16(+/-2) nucleotides of the enzyme-ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5' or 3' end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein-protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.