分子信标检测限制性内切酶活性的新型荧光分析方法

利用分子信标被切割后荧光信号的变化.发展了一种简便的检测限制性内切酶活性的新型荧光分析方法.以限制性内切酶Xsp I为例.将其识别位点嵌入具有茎环结构的分子信标探针(Molecular Beacon,MB)的环状部分,利用这种分子信标在溶液中形成的瞬时二聚体结构,实现了限制性内切酶对分子信标的切割.在优化的条件下,反应初速度与酶的浓度成正比,线性范围为0.05～50 U/mL,检测限为0.05 U/mL.通过改变分子信标环部的识别位点的序列.还可以检测其他限制性内切酶如Alu I的活性.     

分子信标核酸检测技术研究进展

介绍了分子信标设计和分子信标核酸检测原理、技术特性和在基因突变大规模自动化检测中的应用. 分子信标是一种基于荧光共振能量转移现象设计的发卡型寡核苷酸探针,空间结构上呈茎环结构, 环序列是与靶核酸互补的探针,茎序列由与靶序列无关的互补序列构成,茎的一端连上荧光分子,另一端连上淬灭分子.通过空间结构改变决定分子信标发射荧光特性,从而对核酸进行定量检测. 分子信标技术具有操作简单、敏感、特异、可对核酸进行液相实时检测和对活体内核酸动态进行检测等特点,已应用于HIV辅助受体基因等基因突变的大规模自动化检测,是一种新型核酸定量检测技术.     

用于HBV病毒快速诊断的分子信标探针设计

分子信标是一种高灵敏度、高特异性的新型荧光核酸探针.它在与互补DNA或RNA靶序列杂交时放出荧光.利用Genebank中调出已知HBV病毒ayr亚型基因组信息,通过BeaconDesigner4.0软件进行分子信标探针设计,共设计出6条分子信标探针,以便于为目前HBV病毒快速诊断提供参考.     

分子信标在活细胞内的应用

分子信标(molecular beacon,MB)可实时成像观察活细胞内多个特异性靶分子的相互作用,在肿瘤诊断和治疗上有潜在的应用前景。由于核酸酶降解、核酸结合蛋白质结合等非特异性因素,生物稳定性差、转染效率低等成为MB活细胞内应用的瓶颈。稳定的MB并结合高效的转运载体有望成为实时观察活细胞生物学过程及肿瘤诊断和治疗新的工具。     

分子信标用于活细胞内源性mRNA 检测的实验研究

目的:观察分子信标(molecular beacons,MB)在活细胞内及在体水平对内源性m RNA检测的可行性。方法:合成增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,e GFP)的MB,模拟体内条件,分别从MB的稳定性、与目标基因结合的特异性和敏感性进行检测,并通过在活细胞和斑马鱼胚胎内的应用来观察其对内外源性m RNA的检测的可行性。结果:MB在37℃条件下在体外48小时内经琼脂糖凝胶电泳鉴定未见降解,熔解曲线结果显示荧光强度差异无统计学意义(P0.05);并且只有在靶序列存在的条件下MB的信号强度显著增强,MB的信号强度与靶基因浓度呈正相关并能基本稳定存在;经e GFP和靶向e GFP的MB共转染的293T细胞能看到荧光表达,靶向e GFP的MB可特异性结合细胞内e GFP的m RNA;在单细胞期斑马鱼胚胎内共注射MB以及目标单链后能观察到荧光表达。结论:结果表明分子信标可用于活细胞内内源性m RNA的检测。     

抗体Fc片段特异寡核苷酸配基介导的实时定量免疫PCR

目的:利用小鼠IgG抗体Fc片段高特异、高亲和寡核苷酸配基,构建实时定量免疫PCR检测方法,提高抗体检测的灵敏度。方法:用SELEX技术从随机寡核苷酸文库中筛选抗体Fc片段特异寡核苷酸配基,设计合成信标序列,通过不对称PCR法,制备IgG Fc片段的核酸信标配基分子;32P标记核酸信标配基,采用琼脂糖凝胶阻滞双显色法鉴定核酸信标配基与IgG Fc片段结合的亲和力和特异性;制备IgG Fc特异性寡核苷酸信标配基-抗体复合检测分子,构建小鼠IgG Fc片段特异核酸信标配基介导的实时定量免疫PCR检测方法。结果:制备了IgG Fc片段的核酸信标配基分子;凝胶阻滞放射自显影和考马斯亮蓝二次染色结果显示该核酸信标配基分子与IgG Fc片段具有高度亲和力和活性,而且只与非变性IgG结合,与变性IgG不结合;IgG Fc片段的特异核酸信标配基与IgG结合形成复合检测分子,有效完成了信号传递和实时定量PCR信号放大过程。结论:初步建立了一种全新的核酸信标配基介导的免疫PCR检测方法,可有效提高现有IgG类抗体免疫检测的灵敏度和特异性。     

分子信标在DNA生物传感器中的应用

DNA传感器是基于DNA分子相互作用原理设计而成的一种新型的检测技术,具有快速,简单等优点,在基因分析及其他应用领域已显示出越来越重要的价值.分子信标是一种具有发卡式结构的寡核苷酸,由于其能够很好地识别单碱基错配序列,基于发卡式DNA的传感器较传统的单链DNA传感器有更好的检测特异性,目前得到广泛的研究.本文介绍了DNA生物传感器及分子信标的有关原理,并着重介绍了发卡式DNA的结构及其在DNA生物传感器中的应用.     

以分子信标为报告分子的凝血酶检测新方法

以分子信标为报告分子,核酸适体为识别分子,发展了一种新的凝血酶检测方法.含有分子信标互补序列的核酸适体探针与凝血酶结合后,分子信标的荧光信号下降,从而得到凝血酶的浓度信息.该方法快速、灵敏,核酸适体探针无需荧光标记、设计简单,检测限达到0.83nmol/L.     

分子信标芯片计算在0-1整数规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性．而0-1整数规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法．在DNA计算和DNA芯片基础上,提出了基于分子信标芯片解决0-1整数规划问题的DNA计算新模型．与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点,同时指出分子信标芯片技术有望作为新型生物计算的芯片．     

荧光扫描法快速检测肽核酸分子信标标记的单增李斯特菌北大核心CSCD

【目的】建立了单核细胞增多性李斯特菌(Listeria monocytogenes,单增李斯特菌)的肽核酸(Peptide nucleic acid,PNA)分子信标(Molecular beacon)的荧光扫描检测方法,以简化普通PNA原位荧光杂交(Fluorescence in situ hybridization,FISH)检测方法中显微镜观察步骤。【方法】在具有单增李斯特菌特异性的肽核酸探针的5′和3′分别标记报告荧光基团和淬灭基团形成分子信标PNA探针,利用FISH技术和荧光扫描技术对单增李斯特菌进行检测。【结果】用普通PNA探针进行荧光扫描检测时,以N1处理为空白对照,假阳性率11.4%,假阴性率为0;以N2处理为空白时,假阳性率降低至4.3%,但假阴性率上升为18.6%。用分子信标PNA探针进行检测时,用N1为空白对照时,假阳性率8.6%,假阴性率为1.4%;以N2处理作为空白时,假阳性率5.7%,假阴性率为1.4%。与普通探针比较,分子信标PNA探针能有效减少假阳性和假阴性的发生。2种普通PNA探针的杂交成功率分别为83.3%和95.2%;2种"分子信标化"的肽核酸探针的成功率分别为91.7%和90.5%,表明探针两端标记并不会降低与目标菌的杂交成功率。【结论】将液相PNA-FISH和荧光扫描技术结合,通过大通量快速的荧光扫描检测可大幅提高检测效率。同时将肽核酸探针分子信标化,有效的降低了荧光扫描结果的假阳性,并通过了N1和N2两种空白对照处理把假阴性控制在较低的范围。     

Reaction–diffusion wave model for self-assembled network formation of poly(dA)·poly(dT) DNA on mica and HOPG surfaces

Deoxyribonucleic acid (DNA) is a vital molecule for life since it contains genetic information. However, DNA has recently been reported to have unique properties that make it suitable for bionanoelectronic applications, such as the possibility of electrical conductivity and self-organisation. Self-assembled DNA network structures have been observed on several substrates, but the detailed self-assembly mechanism has yet to be determined. The present study investigates self-assembled structures of DNA both theoretically and experimentally. We developed a reaction–diffusion model and used it to investigate pattern formations observed by atomic force microscopy. The computational results qualitatively replicate the network patterns of DNA molecules based on a quantitative agreement with the surface size and timescale. The model can account for the effect of the DNA concentration on pattern formation. Furthermore, peculiar geometric patterns are simulated for mica and highly oriented pyrolytic graphite surfaces.     

5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases

In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.     

表达谱基因芯片

表达谱基因芯片具有高通量、缩微、多参数、平行化等优点.在功能基因组学研究中正发挥越来越重要的作用.就其原理、应用、存在问题及解决策略等进行了综述.     

普通烟草NtODB基因的电子克隆、表达及生物信息学分析

ODB基因在植物同源重组依赖性的DNA双键断裂修复过程中起重要作用,对植物诱变育种具有潜在的应用价值。克隆烟草NtODB基因并分析其表达特征为丰富ODB基因在同源重组DNA修复过程中的作用提供证据。为得到烟草NtODB基因序列,采用电子克隆技术获得该基因cDNA序列并克隆验证。进一步使用生物信息学方法分析该基因表达特征,对预测蛋白的理化性质、信号肽、高级结构等进行预测。生物信息学分析结果表明,NtODB基因开放阅读框包含579个碱基,蛋白含192个氨基酸残基,NtODB蛋白具有碱性和亲水性,主要定位于细胞质内;实时荧光定量PCR检测结果显示NtODB基因在不同组织中呈现组成型表达特征;亚细胞定位检测提示NtODB主要表达于细胞膜和叶绿体。NtODB基因的克隆与表达分析及其蛋白高级结构和理化性质的预测,可为进一步丰富ODB基因在同源重组依赖的DNA修复系统中的作用机制提供证据。     

Computer tomography of the brain in Hamilton.

Computer tomography, a new noninvasive, rapid and easily tolerated technique of brain examination, has been evaluated by analysis of 1000 examinations. It is much more sensitive than conventional radiographic techniques and can resolve soft-tissue structures that differ only slightly in density. It also provides direct visualization of the ventricular system. The range of clinical applications is wide; it is especially useful in differentiating intracerebral hemorrhage from infarction, and in demonstrating many brain tumours, particularly supratentorial, though enhancement with a water-soluble contrast medium injected intravenously is often necessary.     

Structural and Molecular Biology of the eye Lens Membrane

Abstract

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

额外的错配碱基提高T4 DNA连接酶等位特异性连接

连接是一种主要的DNA处理过程。由于较低的商业成本以及核酸底物识别的灵活性,T4 DNA连接酶被广泛应用于生物分子工程,特别是特定核酸序列的等位特异性连接检测。本文评估了在T4 DNA连接酶介导的连接反应中,引入额外的错配碱基对所产生的影响。设计了超过150组DNA/DNA或DNA/RNA带有的额外错配碱基对的组合。结果发现,引入额外的错配碱基对后,T4 DNA 连接酶在DNA/DNA连接中特异性可提高60倍以上,而在DNA/RNA连接中特异性只能提高2倍。在等位特异性连接中,有的错配碱基对可使T4 DNA连接酶的特异性提高600多倍。     

蛋白质复合体及蛋白质相互作用研究新策略—化学交联结合质谱分析法

近年来化学交联法结合质谱分析法被广泛用于蛋白质复合体结构及蛋白质相互作用的研究。研究表明这两种方法的有机结合为研究蛋白质复合体结构及蛋白质相互作用提供了一条新的途径。文章对不同类型的化学交联剂、质谱分析中的Bottom-up 与Top-down 两种研究策略,以及化学交联法结合质谱分析法在蛋白质复合体结构、蛋白质相互作用研究中的应用进行综述。这两种方法的不断发展与完善,将会极大促进生物大分子复合体结构及蛋白质相互作用的研究。     

RecQ helicases: at the heart of genetic stability

The checkpoint-mediated control of DNA replication is essential for maintaining the stability of the genome and preventing cancer in humans. The RecQ family of helicases has been shown to be important for the maintenance of genomic integrity in organisms ranging from bacteria to man. We propose that the RecQ homologue, Sgs1p, has an important function in the S-phase checkpoint response of budding yeast, where it may be both a 'sensor' for damage during replication and a 'resolvase' for structures that arise at paused forks. RecQ helicases may serve a unique function that integrates checkpoint proteins with the recombination and replication fork machinery.