用石英晶体DNA传感器检测单链DNA的探索

利用自组装法,将5′末端标记有巯基醇的单链DNA探针,固定在4.43MHzAT切石英晶体的镀金表面,制成石英晶体DNA传感器,并用该DAN传感器进行了互补单链DNA定量定性检测的探索。     

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.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

古DNA单链测序文库的建立与检测北大核心CSCD

2005年问世的第二代测序技术在古DNA领域的应用,突破了第一代测序技术在绝灭或死亡生物全基因组获取手段上的局限。借助古基因组信息,研究者能够从更为系统的实时分子证据角度,解读诸如人类起源、大型绝灭哺乳动物迁移演化、动植物家养驯化以及早期人类社会生活模式等古生物学、遗传学与演化生物学问题。引入第二代测序技术之后,传统的古DNA研究方法及流程得以改变,剔除了原有的实验流程中耗时的分子克隆步骤,引入了与第二代测序技术紧密相关的古DNA单链测序文库构建环节。古DNA单链测序文库的构建,是将古DNA双链模板变性成单链后,通过向单链古DNA两端添加人工DNA片段,将古DNA分子转变成能被测序仪识别的文库分子。针对古DNA分子微量、高度片段化以及普遍存在碱基损伤的特点,古DNA单链测序文库,能够高效获取古DNA材料中的遗传信息。本文系统介绍古DNA单链测序文库建立流程,以及对文库质量进行检测的方法,为研究者运用第二代测序技术测定绝灭或死亡生物全基因组提供方法借鉴。     

激光、血卟啉对肿瘤细胞DNA单链断裂及其重接的影响

用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。     

基于单链DNA开关调控的多功能生物电路

合成生物电路在生物传感及生物计算方面成为了广泛应用的工具。工程化生物电路系统具有良好的灵活性,同时也具备模块化的特征。在本文中,研究了基于单链DNA开关调控的多功能生物电路的构建方法。通过将计算机辅助设计的单链DNA开关作为核心控制元件,并利用长度为20 bp的toehold区域来激活单链DNA开关,驱动了简单的单向式、循环式以及级联的多层次的生物电路系统。在级联式电路系统中,通过调整单链DNA开关的结构,使信噪比从2.996变成5.274。同时,单链DNA开关作为长单链DNA（784 bp）的一部分,在无细胞蛋白质系统中实现了基因表达调控。因此,本文研究的工程化方法为今后复杂的人工生物电路的构建提供了坚实的技术基础。     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

腾冲嗜热菌单链 DNA 结合蛋白 SSB2 和 SSB3 新的单链 DNA 结合特性

摘要：【目的】揭示腾冲嗜热菌中两个单链DNA结合蛋白SSB2和SSB3的全新的底物结合功能及其不同的体内表达模式。【方法】利用腾冲嗜热菌复制起始位点附近的长度较短的单链DNA为底物,采用非变性聚丙烯酰胺凝胶电泳及western blot方法,研究SSB2和SSB3体外单链DNA结合特征和体内表达模式。【结果】SSB2 与35nt的复制起始区单链DNA（ssDNA）结合, 形成单个SSB2-DNA复合物;当与59 nt ssDNA结合时,可以随着蛋白浓度的递增形成一个或两个SSB2-DNA复合物;而与70n     

DNA氧化性损伤与端粒缩短

末端复制问题（the end replication problem）不能完全解释端粒在某些细胞分裂过程中迅速缩短的现象.40％的高压氧下细胞传代次数降低,端粒缩短速率增大,细胞出现衰老特征,端粒DNA上单链断裂积累.推测端粒缩短的主要原因在于衰老过程中或氧胁迫下端粒DNA单链断裂增多,使端粒末端单链片段在DNA复制时丢失.端粒酶和活性氧对端粒长度的正负调控作用的准确机制还有待于更深入的研究.     

一种有效的DNA序列测定方法

比较了单链和双链DNA(ss和dsDNA)模板和两种不同的聚丙烯酰胺凝胶电泳对DNA序列测定的影响。结果表明:在相同的条件下,ssDNA模板明显优于dsDNA模板;缓冲液梯度聚丙烯酰胺凝胶电泳测定DNA序列的长度在40cm长的胶上可达350nt。而一般的线性聚丙烯酰胺凝胶电泳才可测150nt。对于基因组序列分析,采用单链DNA模板和缓冲液梯度聚丙烯酰胺凝胶电泳是一种有效的测定方法。     

Sensitivity enhancement of DNA sensors by nanogold surface modification

A novel amplified microgravimetric gene sensing system was developed using quartz crystal microbalance modified by gold nanoparticles anchored on its 1,6-hexanedithiol modified gold electrode surface, and ultrasensitive detection of DNA hybridization was accomplished at the level of at least 2 x 10(-16) M.     

A lipid-based method for the preparation of a piezoelectric DNA biosensor

A piezoelectric DNA biosensor was prepared by immobilizing DNA probes on a quartz crystal microbalance (QCM) using a lipid-based method. A QCM electrode was coated with a hybrid bilayer membrane composed of an octadecanethiol monolayer and a lipid monolayer containing biotinylated lipids to establish biotin groups on the electrode surface. A DNA biosensor was prepared by sequentially immobilizing avidin and the biotinylated probe. The DNA biosensor was stable throughout repeated surface regeneration and showed higher sensitivity than that prepared by the conventional chemical method using diimide. We also optimized the surface regeneration conditions and flow rate for flow injection analysis.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Electrochemical study of the interaction between cytochrome c and DNA at a modified gold electrode

The direct electron transfer of surface-confined horse heart cytochrome c (Cyt c) was achieved using COOH-terminated alkanethiolate-modified gold electrode. Later DNA was immobilized on the two-layer modified electrode. The quantitative determination of DNA was explored and the interaction between cytochrome c and DNA was studied. The binding site sizes were determined to be 15 bp per Cyt c molecule with double-stranded (ds) DNA and 30 nucleotides binding one Cyt c molecule with single-stranded (ss) DNA. At the dsDNA/Cyt c/MUA/Au electrode, the rate constant of oxidation electron transfer k(s,ox)=1.59x10(-3)cms-1 was obtained, at the ssDNA/Cyt c/MUA/Au electrode, the value was 2.43x10(-3)ms-1 when the scan rate was 1.0V/s. The different electrodes were characterized with electrochemical quartz crystal microbalance and atomic force microscope.     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

alpha-Lipoic acid and glutathione protect against the prooxidant activity of SOD/catalase mimetic manganese salen derivatives

Manganese(III) N,N'-ethylenebis(salicylideneiminato) chloride (Mn-salen chloride) and manganese(III) N,N'-ethylenebis(3-methoxysalicylideneiminato) chloride (Mn-(3,3'-MeO)salen chloride) are in vitro superoxide dismutase and catalase mimetics. They protect against free radical-related disease in animals, but Mn-salen can also be a potent prooxidant, damaging free DNA. Mn-salen protects human fibroblast DNA against hydrogen peroxide damage, however, damage to free DNA was confirmed by the comet assay. The DNA-damaging activity was dramatically reduced by co-administration with glutathione with the combination being less damaging to free DNA than either molecule alone. alpha-Lipoic acid, an antioxidant disulfide commonly used as a dietary supplement, also prevented Mn-salen prooxidant activity. Mn-(3,3'-MeO)salen protected fibroblasts against hydrogen peroxide as efficiently as Mn-salen and showed little damaging activity against free DNA. Protection was invested by both complexes in the presence and in the absence of EDTA, a potential competing chelator. Stabilities of the complexes with respect to decomposition and inactivation were studied by spectroscopic and electrochemical techniques. The complexes' binding to, and cleavage of, DNA was measured using a quartz crystal resonant sensor. Mn-salen was shown to bind strongly to DNA, prior to cleaving it; Mn-(3,3'-MeO)salen bound weakly and left DNA intact. Co-administration of either glutathione or alpha-lipoic acid appears to inhibit binding by Mn-salen thus preventing DNA-cleavage.     

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions

In recent years, there has been a rapid growth in the number of scientific reports in which the quartz crystal microbalance (QCM) technique has played a key role in elucidating various aspects of biological materials and their interactions. This article illustrates some key advances in the development of a special variation of this technique called quartz crystal microbalance with dissipation monitoring (QCM-D). The main feature and advantage of QCM-D, compared with the conventional QCM, is that it in addition to measuring changes in resonant frequency (Δf), a simultaneous parameter related to the energy loss or dissipation (ΔD) of the system is also measured. Δf essentially measures changes in the mass attached to the sensor surface, while ΔD measures properties related to the viscoelastic properties of the adlayer. Thus, QCM-D measures two totally independent properties of the adlayer. The focus of this review is an overview of the QCM-D technology and highlights of recent applications. Specifically, recent applications dealing with DNA, proteins, lipids, and cells will be detailed. This is not intended as a comprehensive review of all possible applications of the QCM-D technology, but rather a glimpse into a few highlighted application areas in the biomolecular field that were published in 2007.