Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Background: Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results: A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 10(5) and 1.7 x 10(5) per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion: The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide Ligation assay on DNA chips

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

DNA结构的扫描探针显微学研究

1．利用原子力显微镜，我们研究了DNA与邻菲咯琳钻之间的反应。乙醇溶液中DNA复合物的形貌改变很好地说明了静电力在引起DNA凝集中的重要地位。随着醇浓度增加，对应更低的介电常数，静电反应增强。当介电常数降低，平衡离子就会在DNA的磷酸骨架上聚集，并中和负电荷到能引起凝集的程度。2，为了深入认识DNA-表面活性剂复合物的形貌特征，我们对阳离子表面活性剂CTAB引起的DNA凝集体进行了研究。由环状凝集体的体积分析，我们认为DNA发生单分子凝集。除环状体之外，许多不规则的环状体有助于说明DNA环状体的形成过程。部分绕转体和核小体状环状体对应于绕转模型而棒状中间体显示DNA分子也可能采取棒状体到环状体的转化模型。3．DNA分子在形成稳定的凝集体之前会经过一系列的中间体。因为温度是DNA结构的一个重要的决定因素，在其他凝集条件一定时，我们观察了DNA凝集中间体在温度升高时的结构转变。AFM分析得出质粒DNA pBR322的凝集结构受温度的影响很大。低温时，单个分子花束体是DNA分子采取的主要凝集结构。较高温度时，分子变得舒展，但链上有许多可能利于多分子凝集的小环。继续升高温度，DNA分子大范围地发生分子间的单中心、交联。4．在Mg2+，Ca2＋，Sr2和Ba2+分别作为DNA在云母表面的平衡离子时，我们观察了DNA分子在云母表面扩散平衡或被动力俘获的形貌特征。末端距和伸展长度可由原子力显微镜的数据分析结果得到。实验结果表明当Mg2＋，Ca2+和Sr2+为平衡离子时，DNA分子能够在云母表面扩散平衡。然而Ba2+作用时，严重交接的DNA结构说明DNA分子不易在云母表面反应平衡，俘获的程度也随实验条件而改变。在醇溶液中，我们还观察了B-A构象转变对平衡离子种类的依赖关系。四种碱土金属引起B-A构象转变的能力不同，其中Sr2-导致B-A构象转变的程度最大。

DNA、纳米微粒及其相互作用的扫描探针显微学研究

1．采用改进的蒸发液滴中流体毛细流动法，用于展开和固定适于AFM研究的大环状DNA。对长达148.9 kbp的人类基因组3号染色体上一个完整的酵母染色体DNA分子和pB孙22质粒DNA进行了展开固定研究，长度测定显示偏差小于3.5％，结果优于其它展开线性DNA的方法。对其相应的展开机理进行了初步探讨：利用AFM考察了不同长度的DNA分子在云母基底上的分子级分形图案。实验证明DNA分子级的分形图案与DNA的浓度、长度和其它辅助因素相关。我们认为DLA理论或许更适合描述DNA分子在云母基底上形成不同分形图案的体系。2．我们利用阳离子的桥合作用，将质粒DNA固定并展开在云母基底上制得了DNA网络结构。结合实验数据，对质粒DNA形成的机理和可控的参数：离子种类、离子浓度、网孔大小和网孔高度进行了讨论，提出了相应的模型进行解释。我们在半透明的云母基底以及玻璃和蓝宝石等透明基底上构建了不同结构的DNA网络。结合实验数据，讨论了在各种固体基底上形成DNA网络的影响因素，我们认为，固体基底表面的亲水性对于构建DNA网络起重要作用。我们通过控制乙醇的浓度与温度结合原子力显微镜样品定位技术发现，云母基底上形成DNA膜的DNA分子是否移动与处理DNA样品的乙醇浓度有直接关系。对于纯乙醇溶液无论是高温（6O℃）或常温，均未引起DNA分子的移动（无论是吸附在云母基底上的，还是吸附在DNA分子链上的）；如果用混合有水的乙醇溶液（无论其中水的含量多少），不管是高温（60℃）或常温，都会引起DNA膜结构发生变化，即DNA分子发生了移动。热乙醇水溶液对DNA分子的作用比常温情况下更明显1可能是热力学上的因素导致的。3．通过控制银纳米微粒在云母基底上的聚集行为，而非直接通过化学合成的方法，构建了一种新的银结构一纳米银盘。详细阐述了如何得到这种结构的过程，通过原子力显微镜、紫外可见光光谱和X一射线电子能谱技术等工具对其性质也作了相应的表征。这种由纳米粒子组成的银盘与文献报道的单晶纳米银盘相比具有更大的比表面积，在纳米催化等方面有更广阔的应用前景。通过自聚集方式构建纳米银盘结构的方法，对于人们通常认为在溶液中合成的纳米结构与在各种固体基底上表征的纳米结构是一致的观点提出了新的理解。利用戊二醛试剂与发光的纳米微粒萘酰亚胺通过醛胺加成反应形成酰胺键，组装形成二维发光粒子网络。结合原子力显微镜高度形貌图和光谱数据，发现粒子间形成了两种典型的网络结构即，实心六方堆积和空心六方堆积结构。对此我们提出了相应的模型给予解释。4，利用展开的质粒DNA为模板诱导形成了环状的氯化镁纳米结构。原子力显微镜考察表明，这些纳米结构的高度是6.2±1.3-8.2±1.80nm，长度为1.35±0.18到2.93±0.25um。我们以CTAB包裹18nm纳米金和3.5nm纳米银使之形成带正电荷的外壳，利用。NA磷酸骨架带负电荷特性，通过静电自组装方式形成了金属化的纳米网络结构。通过AFM、UV-vis光谱和XPS谱的表征说明，带正电荷外壳的纳米金和纳米银被DNA模板高度地组装形成有序的二维网络结构。5．利用自制的样品定位系统，重复性地对整个基底范围内的样品实行定位，定位的精确度达400nm。这种方法对于样品旋转角度或取出后进行进一步处理都适用。该定位方法依赖于一台个人电脑，一个样品定位仪，一个CCD相机和一套可视光学系统，用于监测透明或半透明基底如，云母、玻璃、蓝宝石、石英和钦酸银等原子力显微镜广泛使用的基底表面或背面特征。作为对这种定位方法的应用，我们用不同的灯M操作模式，不同的针尖对单个的DNA分子、单个的DNA-蛋白质复合物和DNA网络在样品移动或拿出样品台后进行了定位实验。这种方法的精度和分辨率，对于一般的商用或自制SPM（AFM,STM，sNOM）系统都可以适用。

DNA-聚苯胺体系的电化学及扫描探针显微学研究

DNA-聚苯胺复合物在低维纳米材料制作、生物传感器、DNA杂交反应检测以及生物芯片开发等方面具有潜在的应用价值。鉴于此，我们在本论文中着重研究了以下两方面内容：1．利用循环伏安法研究了DNA的存在对聚苯胺电化学行为的影响。研究发现，虽然，苯胺的循环伏安聚合过程不受DNA的影响，但是DNA的存在会显著降低聚苯胺修饰电极在HCl空白溶液中循环伏安扫描时的峰电流值。初步推测可能是由于DNA分子在聚苯胺膜上的吸附阻碍了电极上电子的传递而造成的。2．通过自组装方法从水溶液中构造出了分散性良好的苯胺-DNA复合物纳米线，并以此为前驱体构造出了以DNA为模板的聚苯胺纳米线。另外，利用改进后的气流展开法可以将所得到的纳米线有序排列到基底上。用原子力显微镜对所得到的纳米线进行了表征，结果显示纳米线形貌规整、排列有序、背景清晰。

Atomic force microscopy and surface-enhanced Raman scattering detection of DNA based on DNA-nanoparticle complexes

We report a simple method for the label-free detection of double-stranded DNA using surface-enhanced Raman scattering (SERS). We prepared cetyltrimethylammonium bromide (CTAB)-capped silver nanoparticles and a DNA-nanoparticle complex by adding silver nanoparticles to lambda-DNA solutions. In the present study, the utilization of CTAB-capped silver nanoparticles facilitates the electrostatic interaction between DNA molecules and silver nanoparticles; at the same time, the introduction of DNA avoids adding aggregating agent for the formation of nanoparticle aggregates to obtain large enhancement of DNA, because the DNA acts as both the probe molecules and aggregating agent of Ag nanoparticles.

DNA based gold nanoparticles colorimetric sensors for sensitive and selective detection of Ag(I) ions

In this work, we reported both unlabeled and labeled sensing strategies for Ag(I) ions detection by using the DNA based gold nanoparticles (AuNPs) colorimetric method. In the unlabeled strategy, C-base riched single strand DNA (C-ssDNA) enwinded onto AuNPs to form AuNPs/C-ssDNA complex. In the labeled method, sulfhydryl group modified C-ssDNA (HS-C-ssDNA) was covalently labeled on AuNPs to produce AuNPs-S-C-ssDNA complex. In both strategies, C-ss DNA or HS-C-ssDNA could enhance the AuNPs stability against the salt-induced aggregation. However, the presence of Ag(I) ions in the obtained AuNPs/C-ssDNA or AuNPs-S-C-ssDNA complex would decrease such stability to display purple even blue colors due to the formation of Ag(I) ions mediated C-Ag(I)-C base pairs. Through this phenomenon, Ag(I) ions could be detected qualitatively and quantitatively using both unlabeled and labeled sensing strategies.

7-Amino actinomycin D bound to single-stranded hairpin DNA enhanced by loop sequence-dependent luminescent Eu3+ and Tb3+ binding

Lanthanide Eu3+ and Tb3+ ions have been widely used in luminescent resonance energy transfer (LRET) for bioassays to study metal binding microenvironments. We report here that Eu3+ or Tb3+ can increase the binding affinity of antitumor antibiotic drug agent, 7-amino actinomycin D (7AACTD), binding to 5'-GT/TG-5' or 5'-GA/AG-5' mismatched stem region of the single-stranded hairpin DNA. Further studies indicate that the effect of Eu3+ or Tb3+ on 7AACTD binding is related to DNA loop sequence. Our results will provide new insights into how metal ions can enhance antitumor agents binding to their targets.

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.

Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy

The hybridization of immobilized oligonucleotides probe strands with solution phase targets is the underlying principle of microarraybased techniques for the analysis of DNA variation. To study the kinetics of DNA/DNA hybridization, target DNA is often prior labeled with markers. A label-free method of electrochemical impedance spectra (EIS) for study the hybridization in process was reported. The Langmuir model was used to determine the association rate constant (K-on), the dissociation rate constant (K-off) and the affinity rate constant (K-A), for perfect matched DNA hybridization. The results show that, EIS is a successful technique possessing high effectivity and sensitivity to study DNA/DNA hybridization kinetics. This work can provide another view on EIS for the studying of DNA/DNA hybridization.

Reversible B/Z-DNA transition under the low salt condition and non-B-form polydApolydT selectivity by a cubane-like europium-L-aspartic acid complex

We report here that a cubane-like europium-L-aspartic acid complex at physiological pH can discriminate between DNA structures as judged by the comparison of thermal denaturation, binding stoichiometry, temperature-dependent fluorescence enhancement, and circular dichroism and gel electrophoresis studies. This complex can selectively stabilize non-B-form DNA polydApolydT but destabilize polydGdCpolydGdC and polydAdTpolydAdT. Further studies show that this complex can convert B-form polydGdCpolydGdC to Z-form under the low salt condition at physiological temperature 37 degrees C, and the transition is reversible, similar to RNA polymerase, which turns unwound DNA into Z-DNA and converts it back to B-DNA after transcription. The potential uses of a left-handed helix-selective probe in biology are obvious. Z-DNA is a transient structure and does not exist as a stable feature of the double helix. Therefore, probing this transient structure with a metal-amino acid complex under the low salt condition at physiological temperature would provide insights into their transitions in vivo and are of great interest.

The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.

Growth of cationic lipid toward bilayer, lipid membrane by solution spreading: scanning probe microscopy study

The growth of cationic lipid dioctadecyldimethylammonium bromide (DODAB) toward bilayer lipid membrane (BLM) by solution spreading on cleaved mica surface was studied by atomic force microscopy (AFM). Bilayer of DODAB was formed by exposing mica to a solution of DODAB in chloroform and subsequently immersing into potassium chloride solution for film developing. AFM studies showed that at the initial stage of the growth, the adsorbed molecules exhibited the small fractal-like aggregates. These aggregates grew up and expanded laterally into larger patches with time and experienced from monolayer to bilayer, finally a close-packed bilayer film (5.4 +/- 0.2 nm) was approached. AFM results of the film growth process indicated a growth mechanism of nucleation, growth and coalescence of dense submonolayer, it revealed the direct information about the film morphology and confirmed that solution spreading was an effective technique to prepare a cationic bilayer in a short time.

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.

Investigation of the influence on conformational transition of DNA induced by cationic lipid vesicles

Recent studies have focused on the structural features of DNA-lipid assemblies. In this paper we take nile blue A (NBA) as a probe molecule to study the influence of the conformational transition of DNA induced by didodecyldimethylammonium bromide (DDAB) cationic vesicles to the interaction between DNA and the probe molecules. We find that upon binding to DNA, a secondary conformational transition of DNA induced by the cationic liposome from the native B-form to the C-form resulted in the change of binding modes of NBA to DNA and different complexes are formed between DNA, DDAB and NBA.