Damage of DNA ends induced by mechanical force during AFM nano-manipulation

An experimental and statistical study was carried out to explore the effects of mechanical forces on the ends of linear double-stranded DNA (dsDNA) fragments. Mechanical force was applied onto individual DNA molecules during atomic force microscope (AFM)-based picking-up manipulation. By comparing the PCR efficiency of two DNA fragments with primers either at ends or at the inner regions, it was found that the ends of DNA fragments were damaged during picking-up process.     

蚕沙叶绿素铜配合物与DNA的相互作用

文章从蚕沙中提取出叶绿素(CHL),并由此制备得到了蚕沙叶绿素铜配合物(Cu-CHL)。采用琼脂糖凝胶电泳实验研究了在白炽灯光照下Cu-CHL对pBR 322 DNA的切割能力。实验结果表明铜叶绿素对pBR 322 DNA具有光断裂能力,最佳断裂浓度范围为178.5～357μM。并采用紫外光谱、CD光谱和粘度法探讨了二者的结合模式,结果显示其为外部结合模式,结合常数为2.04×106m-1。     

生物大分子在纳米微孔中迁移运动的研究进展

DNA等带电生物大分子通过各种生物膜上的纳米微孔的迁移是生命现象中的关键运动步骤之一,对这种迁移过程的研究不仅有助于了解生命运动的分子机理,在快速DNA测序、基因治疗和药物传输的控制等方面也有潜在应用价值。本文介绍了近年来关于DNA分子通过纳米微孔迁移过程在实验测定、理论分析和计算机模拟等方面的研究进展,重点关注迁移动力学及其机理,特别是迁移时间和分子链长间的标度关系等。     

Capacitance DNA bio-chips improved by new probe immobilization strategies

Label-free DNA detection plays a crucial role in developing point-of-care biochips. Capacitance detection is a promising technology for label-free detection. However, data published in literature often show evident time drift, large standard deviation, scattered data points, and poor reproducibility. To address these problems, mercapto-hexanol or similar alkanethiols are usually considered as blocking agents. The aim of the present paper is to investigate new blocking agents to further improve DNA probe surfaces. Data from AFM, SPR, florescence microscopy, and capacitance measurements are used to investigate new lipoate and ethylene-glycol molecules. The new surfaces offer further improvements in terms of diminished detection errors. Film structures are investigated at the nano-scale to justify the detection improvements in terms of probe surface quality. This study demonstrates the superiority of lipoate and ethylene-glycol molecules as blocking candidates when immobilizing molecular probes onto spot surfaces in label-free DNA biochip.     

A Single Molecular Beacon Probe Is Sufficient for the Analysis of Multiple Nucleic Acid Sequences

Molecular beacon (MB) probes are dual‐labeled hairpin‐shaped oligodeoxyribonucleotides that are extensively used for real‐time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real‐time assays and improve the accuracy of single‐nucleotide polymorphism genotyping.     

Quantum‐Dot‐Functionalized Poly(styrene‐co‐acrylic acid) Microbeads: Step‐Wise Self‐Assembly,Characterization, and Applications for Sub‐femtomolar Electrochemical Detection of DNA Hybridization

A novel nanoparticle label capable of amplifying the electrochemical signal of DNA hybridization is fabricated by functionalizing poly(styrene‐co‐acrylic acid) microbeads with CdTe quantum dots. CdTe‐tagged polybeads are prepared by a layer‐by‐layer self‐assembly of the CdTe quantum dots (diameter = 3.07 nm) and polyelectrolyte on the polybeads (diameter = 323 nm). The self‐assembly procedure is characterized using scanning and transmission electron microscopy, and X‐ray photoelectron, infrared and photoluminescence spectroscopy. The mean quantum‐dot coverage is (9.54 ± 1.2) × 10³ per polybead. The enormous coverage and the unique properties of the quantum dots make the polybeads an effective candidate as a functionalized amplification platform for labelling of DNA or protein. Herein, as an example, the CdTe‐tagged polybeads are attached to DNA probes specific to breast cancer by streptavidin–biotin binding to construct a DNA biosensor. The detection of the DNA hybridization process is achieved by the square‐wave voltammetry of Cd²⁺ after the dissolution of the CdTe tags with HNO₃. The efficient carrier‐bead amplification platform, coupled with the highly sensitive stripping voltammetric measurement, gives rise to a detection limit of 0.52 fmol L^?1 and a dynamic range spanning 5 orders of magnitude. This proposed nanoparticle label is promising, exhibits an efficient amplification performance, and opens new opportunities for ultrasensitive detection of other biorecognition events.     

Cationic Polyelectrolyte Amplified Bead Array for DNA Detection with Zeptomole Sensitivity and Single Nucleotide Polymorphism Selectivity

A highly sensitive strand specific DNA assay, which consists of a peptide nucleic acid (PNA) probe, a cationic conjugated polymer ( PFVP ), and self‐assembled polystyrene beads in microwell arrays on silicon chip, is reported. PFVP , as an efficient signal amplifier and signal reporter, has been specially designed and synthesized to be compatible with commercial confocal microscopes for sensing on solid substrates. The assay operates on the net increase in negative charge at the PNA surface that occurs upon single‐stranded DNA hybridization, which subsequently allows complex formation with the positively charged PFVP to favor energy transfer between the polymer and Cy5‐labeled target. With maximized surface contact provided by bead arrays and signal amplification provided by PFVP , this assay allows detection of ～300 copies of Cy5‐labeled DNA using a commercial confocal microscope. In addition, the same strategy is also extended for label‐free DNA detection with a detection sensitivity of 150 attomole. Excellent discrimination against single nucleotide polymorphism (SNP) is also demonstrated for both Cy5‐labeled and label‐free target detection. This study indicates that cationic conjugated polymers have great potential to be incorporated into the widely used microarray technology for simplified process with improved detection sensitivity.     

DNA与（S）-2-（5-氟尿嘧啶-1-基-乙酰基）氨基-1，4-丁二酸相互作用的电化学和光谱学研究

以自组装制得的DNA修饰电极为工作电极,采用循环伏安研究了抗癌药物（S）-2-（5-氟尿嘧啶-1-基-乙酰基）氨基-1,4-丁二酸（5FUASP）与DNA的相互作用,还结合紫外-可见光谱进一步研究了这种相互作用。循环伏安测试结果表明：5FUASP与DNA在pH=6．86的缓冲溶液中有强烈的相互作用,在电极表面反应的过程为准可逆电化学反应一化学反应偶合（Ec）过程。当扫描速度较低时,EC反应是扩散控制过程;紫外吸收光谱表明抗癌药物5FUASP与DNA通过插入作用相互结合。     

质粒剂量和免疫次数对基因枪免疫DNA疫苗免疫反应的影响

目的研究质粒剂量和免疫次数对基因枪免疫DNA疫苗免疫反应的影响。方法构建携带HIV-1 Env gp145基因的DNA疫苗质粒p1.0-Env,分别采用肌肉注射和基因枪方法免疫BALB/c小鼠。其中,基因枪免疫采用不同剂量或不同次数。ELISA法检测小鼠体内Env特异性抗体水平。结果基因枪免疫小鼠诱导的抗体水平显著高于肌肉注射免疫。在0.1～2.25μg剂量范围内,基因枪免疫小鼠体内的Env特异性抗体水平随疫苗剂量的增加不断提高。加大免疫剂量至5μg和10μg,不能提高体液免疫反应水平。基因枪免疫1次和2次所诱导的特异性抗体水平很弱,免疫3次可诱导高水平的抗体应答。结论基因枪免疫能有效提高DNA疫苗所诱导的抗体应答,小鼠的最适免疫剂量约为2.25μg,2.25μg免疫3次可诱导较强的体液免疫反应。