时间分辨荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制整合剂异硫氰酸苯基-EDTA将铕离子标记连接于链霉亲和素分子中,通过光化学反应制备生物素标记pUC118DNA探针,与固定在聚苯乙烯微滴板中的靶DNA杂交后,以铕离子Eu(3+)标记的链霉亲和素为检测物,检测靶DNA的含量,可检测到30pg的靶DNA．     

DNA提取方法的研究进展

样本DNA的提取是PCR反应的关键步骤。本文介绍了近年来国内外几种常用的DNA提取方法,如：传统法、螯合树脂法、玻璃粉法、磁珠法、免疫亲和法等,并对各种方法的优缺点进行了综合评述。     

基于亲和探针的药物靶点鉴定技术研究进展

药物靶点的鉴定和相关研究在药学研究领域具有重要的理论指导意义和实用价值。利用亲和探针偶联靶分子的方法是目前发现药 物靶点的主要手段之一。该方法可从分子水平发现药物的作用靶点,从而对药物的分子作用机制提供细胞水平的直接证据。从 DNA 和小 分子药物探针的构筑和应用入手,对近些年鉴定 DNA 损伤识别蛋白的研究进展进行了较为详尽的讨论,并简要介绍目前探索小分子药物 作用靶点的主流技术。作为亲和偶联鉴定药物作用靶点方法的重要组成部分,亲和探针设计的合理性关系到方法本身的可操作性以及鉴 定结果的可靠性。从多个角度对 DNA 探针和小分子药物探针的设计经验进行了较为系统的总结,例如经典的亲和纯化分离方法,以及更 为高效的光激发共价偶联技术等。这些方法和思路为探索 DNA 损伤相关蛋白质的功能以及小分子药物的细胞作用机制提供了丰富的研究 工具,有助于从分子水平理解药物的作用机制。     

用磁珠富集法从AFLP片段中分离微卫星DNA标记

将花生(Arachis hypogaea L.)基因组DNA经酶切后转变成AFLP DNA片段,然后用生物素标记的简单重复序列(SSR)作探针与其杂交,杂交复合物固定到包被有链亲和素的磁珠上,经过一系列的洗涤过程,含有SSR的AFLP片段被吸附在磁珠表面.这些片段经洗脱下来后,先用对应的AFLP引物扩增,再进行克隆和测序,根据SSR两端的保守序列设计引物,经过多态性分析后,便可得到微卫星DNA标记.整个实验过程操作简单、消耗少,可在一周内完成,可作为从植物中分离SSR的一种简单有效的方法.     

用磁珠富集法从AFLP片段中分离微卫星DNA标记

将花生(Arachis hypogaea L．)基因组DNA经酶切后转变成AFLP DNA片段,然后用生物素标记的简单重复序列(SSR)作探针与其杂交,杂交复合物固定到包被有链亲和素的磁珠上,经过一系列的洗涤过程,含有SSR的AFLP片段被吸附在磁珠表面。这些片段经洗脱下来后,先用对应的AFLP引物扩增,再进行克隆和测序,根据SSR两端的保守序列设计引物,经过多态性分析后,便可得到微卫星DNA标记。整个实验过程操作简单、消耗少,可在一周内完成,可作为从植物中分离SSR的一种简单有效的方法。     

艾氏腹水癌患鼠腹水中一种高分子量DNA结合蛋白的分离

 本文利用免疫吸收法和免疫亲和层析法,从艾氏腹水癌患鼠腹水DNA结合蛋白中,分离得到了一种高分子量DNA结合蛋白。在免疫双扩散反应中,它与抗艾氏腹水癌患鼠血清DNA结合蛋白的兎抗血清反应形成一条沉淀线,但与正常小鼠血清DNA结合蛋白的兎抗血清不形成沉淀线。该DNA结合蛋白样品用2-巯基乙醇还原后,经SDC-PAGE分析,测得其分子量约为41000。     

正常人和SLE患者血清中抗DNA抗体的研究

用亲和层析法对6例正常人和4例SLE患者血清中抗DNA抗体进行提取和定量研究,发现正常人血清中抗DNA抗体的组成IgM/IgG大于1,是以IgM为主的抗体,SLE患者血清中抗DNA抗体含量高,IgM/IgG小于1,是以IgG为主的抗体。用胰蛋白酶降解提取的抗DNA抗体再借助于SDS—聚丙烯酰胺凝胶电泳对正常人和SLE患者抗DNA抗体的结构进行初步探讨,电泳结果表明正常人和SLE患者纯化抗DNA抗体经胰蛋白酶降解以后,正常人在52.2Kd区有一特异性降解片段,而SLE患者则在33.6Kd区有明显的降解片段富集,且表明它们是抗DNA—IgG所产生的。这说明SLE患者血清中抗DNA—IgG不仅在数量上比正常人有所增加,而且在结构上也有所不同。此外,这两种不同的抗DNA—IgG被胰蛋白酶降解的速度也有差异。     

DNA连接酶Ⅲ在线粒体基因组完整性保持中的作用

真核DNA连接酶(DNA ligase)通过催化ATP依赖的双链DNA切口连接而在DNA复制、重组和修复过程中发挥了重要作用．DNA连接酶Ⅲ(Lig3)是一种独特性的连接酶,既可定位于细胞核,又可定位于线粒体．Lig3通过与DNA修复蛋白XRCC1作用而参与了碱基切除修复和其他单链断裂修复．但Lig3以XRCC1不依赖方式在线粒体DNA完整性保持方面发挥了更为重要的作用．这些研究为Lig3功能和DNA修复研究提供了新的视野．     

时间分辩荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制螯合剂异硫氰酸苯基ＥＤＴＡ将希有铕离子标中接于链霉亲和素分子中,通过光化学反应制备生物系标记ＰＵＣ１１８ＤＮＡ探针,与固定在聚苯乙烯微滴板中的靶ＤＮＡ杂交后,以铕离子Ｅｕ＾３＋标边霉亲和素为检测物,检测靶ＤＮＡ的含量,可检测到３０ｐｇ的靶ＤＮＡ。     

一种蜘蛛基因组DNA的简易提取方法

本文介绍了1种改进的蜘蛛基因组DNA的提取方法.通过与传统DNA提取方法的比较,本方法具有可在常温条件下进行、DNA得率高、简便、经济等优势.     

纳米粒子标记DNA 探针在电化学DNA 生物传感器中的应用

介绍了纳米电化学DNA生物传感器的基本概念和分类,并介绍了用于DNA标记的纳米粒子的六种类型及其三大检测方法,在此基础上对纳米电化学DNA生物传感器在基因检测、疾病诊断、DNA检测等方面的最新进展进行了综述与讨论。     

Phase Transition and Optical Properties of DNA–Gold Nanoparticle Assemblies

We review recent work on DNA-linked gold nanoparticle assemblies. The synthesis, properties, and phase behavior of such DNA–gold nanoparticle assemblies are described. These nanoparticle assemblies have strong optical extinction in the ultraviolet and visible light regions; hence, the technique is used to study the kinetics and phase transitions of DNA–gold nanoparticle assemblies. The melting transition of DNA–gold nanoparticle assemblies shows unusual trends compared to those of free DNA. The phase transitions are influenced by many parameters, such as nanoparticle size, DNA sequence, DNA grafting density, DNA linker length, interparticle distance, base pairing defects, and disorders. The physics of the DNA–gold nanoparticle assemblies can be understood in terms of the phase behavior of complex fluids, with the colloidal gold interaction potential dominated by DNA hybridization energies.     

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ～1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen–phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.     

抗肿瘤多药耐药纳米粒的制备及其对MCF-7/ADR 细胞的体外评价

目的:肿瘤的多药耐药现象会显著降低肿瘤细胞内药物浓度,本研究通过制备抗肿瘤多药耐药的靶向给药系统来逆转肿瘤的耐药性以提升细胞对药物的敏感性,从而降低该现象对癌症治疗的阻碍。方法:本文使用乳化溶剂挥发法制备以含姜黄素两亲性嵌段共聚物载体、以紫杉醇和磁性粒为核心的抗肿瘤多药耐药纳米粒,使用透射电镜和动态粒径散射仪等对纳米粒进行表征和磁响应性测试后,使用MTT法测定纳米粒对肿瘤耐药细胞MCF-7/ADR的抑制率以探究给药系统的耐药逆转性能。结果:制备的抗肿瘤多耐药纳米粒粒径为105 nm左右,磁响应性良好。所制得载紫杉醇纳米粒包封率为74.74%,载药率为12.40%。纳米粒可以通过磁场和生物素受体介导作用促进肿瘤细胞对粒子的内化,以增加抗癌药物的蓄积。与游离紫杉醇相比,逆转细胞耐药指数达8.5。结论:纳米系统在维持自身稳定性同时,能够凭借协同作用和靶向作用较大程度提升药物对耐药肿瘤细胞的杀伤效果。     

Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid

Lately, the isolation of DNA using magnetic nanoparticles has received increased attention owing to their facile manipulation and low costs. Although methods involving their magnetic separation have been extensively studied, there is currently a need for an efficient technique to isolate DNA for highly sensitive diagnostic applications. We describe herein a method to isolate and purify DNA using biofunctionalized superparamagnetic nanoparticles synthesized by a modified polyol method to obtain the desired monodispersity, followed by surface modification with meso-2,3-dimercaptosuccinic acid (DMSA) containing carboxyl groups for DNA absorption. The DMSA-coated magnetic nanoparticles (DMSA-MNPs) were used for the isolation of DNA, with a maximum yield of 86.16%. In particular, we found that the isolation of DNA using small quantities of DMSA-MNPs was much more efficient than that using commercial microbeads (NucliSENS-easyMAG, BioMérieux). Moreover, the DMSA-MNPs were successfully employed in the isolation of genomic DNA from human blood. In addition, the resulting DNA–nanoparticle complex was directly subjected to PCR amplification without prior elution, which could eventually lead to simple, rapid, sensitive and integrated diagnostic systems.     

Detection of the third and fourth heart sounds using Hilbert-Huang transform

Background

Magnetic nanoparticles are gaining great roles in biomedical applications as targeted drug delivery agents or targeted imaging contrast agents. In the magnetic nanoparticle applications, quantification of the nanoparticle density deposited in a specified region is of great importance for evaluating the delivery of the drugs or the contrast agents to the targeted tissues. We introduce a method for estimating the nanoparticle density from the displacement of tissues caused by the external magnetic field.

Methods

We can exert magnetic force to the magnetic nanoparticles residing in a living subject by applying magnetic gradient field to them. The nanoparticles under the external magnetic field then exert force to the nearby tissues causing displacement of the tissues. The displacement field induced by the nanoparticles under the external magnetic field is governed by the Navier's equation. We use an approximation method to get the inverse solution of the Navier's equation which represents the magnetic nanoparticle density map when the magnetic nanoparticles are mechanically coupled with the surrounding tissues. To produce the external magnetic field inside a living subject, we propose a coil configuration, the Helmholtz and Maxwell coil pair, that is capable of generating uniform magnetic gradient field. We have estimated the coil currents that can induce measurable displacement in soft tissues through finite element method (FEM) analysis.

Results

From the displacement data obtained from FEM analysis of a soft-tissue-mimicking phantom, we have calculated nanoparticle density maps. We obtained the magnetic nanoparticle density maps by approximating the Navier's equation to the Laplacian of the displacement field. The calculated density maps match well to the original density maps, but with some halo artifacts around the high density area. To induce measurable displacement in the living tissues with the proposed coil configuration, we need to apply the coil currents as big as 10⁴A.

Conclusions

We can obtain magnetic nanoparticle maps from the magnetically induced displacement data by approximating the Navier's equation under the assumption of uniform-gradient of the external magnetic field. However, developing a coil driving system with the capacity of up to 10⁴A should be a great technical challenge.     

Mitochondrial DNA Repair Pathways

It has long been held that there is no DNA repair in mitochondria. Early observations suggestedthat the reason for the observed accumulation of DNA damage in mitochondrial DNA is thatDNA lesions are not removed. This is in contrast to the very efficient repair that is seen inthe nuclear DNA. Mitochondrial DNA does not code for any DNA repair proteins, but it hasbeen observed that a number of repair factors can be found in mitochondrial extracts. Mostof these participate in the base excision DNA repair pathway which is responsible for theremoval of simple lesions in DNA. Recent work has shown that there is efficient base excisionrepair in mammalian mitochondria and there are also indications of the presence of morecomplex repair processes. Thus, an active field of mitochondrial DNA repair is emerging. Anunderstanding of the DNA repair processes in mammalian mitochondria is an important currentchallenge and it is likely to lead to clarification of the etiology of the common mutations anddeletions that are found in mitochondria, and which are thought to cause various humandisorders and to play a role in the aging phenotype.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80 °C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.     

Single-molecule enzymology based on the principle of the Millikan oil drop experiment

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.     

Loop‐mediated isothermal amplification of single pollen grains

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field‐enabled, high‐throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop‐mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field‐based, high‐throughput analysis.