DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations.The method is based on the formation and deformation of double-stranded DNA(dsDNA)membranes on a gold surface.It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface.In these systems,Fe(CN)63- was used as the reporter.As the temperature increases,a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears.At a special temperature,the and single base mutation target.Thus,the system provides a simple and sensitive method to detect DNA point mutations without labeling targets.     

双链DNA 膜效应在序列点突变电化学检测中的应用

报道一种基于金表面的双链DNA膜效应检测DNA点突变的新方法。致密的双链DNA分子层可以将电化学信号分子禁闭在金表面和双链DNA之间,或者将信号分子与金表面隔离开,使其无法接触裸金面．在实验系统中采用Fe(CN）6^3-为信号分子,在升高温度时,双链DNA膜被破坏,信号分子离开或接触金表面,解链曲线会出现一个陡峭的电化学信号变化。在特定的温度下,完全互补的序列和单碱基点突变的序列的信号比达到了100：1。这种方法简单而且灵敏,同时避免了复杂的共价修饰信号分子的过程。     

Detection of single-base mismatch at distal end of DNA duplex by electrochemical impedance spectroscopy

Herein, we report an anomalous electrochemical behavior of surface-bound DNA duplex that has single-base mismatches at its distal end. Single-stranded 15-base DNA was immobilized at its 5'end onto gold electrode surfaces. After hybridization with complementary or mismatched DNA, electrochemical impedance spectra were obtained using [Fe(CN)(6)]3-/4- as redox marker ions. Hybridization with the complementary DNA reduced the charge-transfer resistance (R(CT)), whereas single-base mismatches at the distal end of the duplex largely increased the R(CT). This anomaly was found only with the distal end: the increase in R(CT) was not observed for mismatches at either the middle or the proximal end. These results indicate that electrochemical detection of single-base alterations at an end of sample DNA is exceptionally easy because of the diametrically opposite responses. This detection principle is promising for the typing of single-nucleotide polymorphisms in combination with the single-base primer extension protocol.     

Rapid single-base mismatch detection in genotyping for phenylketonuria

Phenylketonuria (PKU) is a metabolic disorder that results from a deficiency of hepatic phenylalanine hydroxylase (PAH). Identification of the PKU genotype is useful for predicting clinical PKU phenotype. More than 400 mutations resulting in PAH deficiency have been reported worldwide. We used a genedetecting instrument to identify the nine prevalent Japanese mutations in the PAH gene among 31 PKU patients as a preliminary study. This instrument can automatically detect mutations through the use of allele-specific oligonucleotide (ASO) capture probes, and gave results comparable to those of sequencing studies. Each country has uniquely prevalent and specific mutations causing PKU, and less than 50 types of such mutations are generally present in each country. Early genotyping of PKU makes it possible to identify the phenotype and select the optimal therapy for the disease. For early genotyping, the instrumental method described here shortens the time required for genotyping based on mRNA and/or genomic DNA of PKU parents.     

Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

G-quadruplex formation by single-base mutation or deletion of mitochondrial DNA sequences

Background

Mitochondrial DNA (mtDNA) mutations could lead to mitochondrial dysfunction, which plays a major role in aging, neurodegeneration, and cancer. Recently, we have highlighted G-quadruplex (G4) formation of putative G4-forming (PQF) mtDNA sequences in cells. Herein, we examine structural variation of G4 formation due to mutation of mtDNA sequences in vitro.

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

新型青霉素生物传感器的研究

以青霉素为模板分子,采用溶胶-凝胶法合成分子印迹膜,以浸泡的方法移除印迹分子,制备青霉素分子印迹膜电极。本印迹电极能有效地避免类似物对其测定的干扰。通过循环伏安法研究传感器对青霉素的响应特性,结果表明:富集时间为200 s,在0.1~1.8μg/L质量浓度范围内,青霉素在磷酸缓冲液(PBS)中的电流强度与其浓度呈良好的线性关系。吸附后的膜电极用甲醇洗脱后再生,可以重复利用3次,可以应用到实际检测中。     

Chemical cleavage of DNA duplexes with single base mismatches as a basis for detection of random point mutations

The most promising approaches to detection of random point mutations are based on chemical cleavage of mismatches and other noncomplementarities. To demonstrate the specificity of this method, a model system was obtained for the first time as sets of 50-mer imperfect DNA duplexes containg all variants of mismatched and unpaired internal residues located in an invariant context and flanked by either A · T or G · C base pairs. Chemical cleavage of DNA duplexes immobilized on magnetic beads via the biotin-streptavidin interaction was accomplished using potassium permanganate or hydroxylamine, which are sensitive to the secondary DNA structure and react with thymine and cytosine, respectively. The reactivity of different mismatches was connected with the local duplex structure and depended on their type, orientation, and flanking nucleotides. The use of potassium permanganate and hydroxylamine to modify a heteroduplex mixture makes it possible to unambiguously detect a mismatch and, based on the type of reagent and the size of the cleavage products, to suppose the type and position of the mismatch and the flanking nucleotides. The model system can be used to evaluate the sensitivity of a chemical cleavage method and to control false-positive and false-negative results when different protocols are applied to the detection of DNA point mutations.     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.     

钩端螺旋体外膜疫苗的流行病学效果评价

为了评价国产钩端螺旋体外膜疫苗的流行病学效果。于1998年6－10月,用队列研究和病例对照研究等方法在钩体病流行区5－60岁农业人口 ,观察比较接种组和对照组钩体发病情况。队列研究显示,钩体外膜疫苗对同血清群钩体的保护率为75．17％,效果指烽为4．03。1：2配对调查的疫苗保护率为81．25％,1：3配对结果为73．33％。用筛选法估计的疫苗效果为755。不同研究方法的结果相近,均一致说明,该疫苗的流行病学效果较理想。研究结果还显示,该疫苗对异血清群钩体也有一定的保护作用。     

Mitochondrial membrane cholesterol,the voltage dependent anion channel (VDAC), and the Warburg effect

Normal cells of aerobic organisms synthesize the energy they require in the form of ATP via the process of oxidative phosphorylation. This complex system resides in the mitochondria of cells and utilizes oxygen to produce a majority of cellular ATP. However, in most tumors, especially those with elevated cholesterogenesis, there is an increased reliance on glycolysis for energy, even in conditions where oxygen is available. This aerobic glycolysis (the Warburg effect) has far reaching ramifications on the tumor itself and the cells that surround it. In this brief review, we will discuss how abnormally high membrane cholesterol levels can result in a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH-7777). We have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution and transport. Work in our laboratory demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

电化学生物传感器快速检测DNA研究进展

本简要地介绍了DNA电化学生物传感器研究的最新进展,重点讨论了改善生物传感器选择性和灵敏度的技术和方法。     

膜技术在生物检测中的应用研究进展

膜具有保护、支撑、分散和分离的功能,通过表面修饰和接枝/负载选择性配基或催化剂等可赋予膜更多的功能,因此膜技术在生物检测中的应用越来越广泛,其发挥作用的方式和途径也极其多样化。对功能膜的理性设计可以满足生物检测过程中不同环节的需求,包括生物样品的前处理、制备、响应和传感等。文中简述了分离膜的功能化方法,并对目前膜技术在样品制备和检测过程中的应用以及多功能膜集成的研究进行综述。通过梳理面向生物检测的功能膜设计、制备和应用研究进展,以期更好地发挥膜材料结构和功能的优势,构建更加"适应"检测环境的高效、稳定检测平台。     

Structural effect of the anticancer agent 6-thioguanine on duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G·T) and opposite cytosine (S6G·C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson–Crick hydrogen bonding in place. The S6G·T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.     

通过替代失活的方法分析核酸片段在肺炎链球菌毒力中的作用

目的鉴定基因spd—ABC、spd-1672、sp-1673和长间隔序列spd—J在肺炎链球菌（Streptococcus pneumoniae,S．pn）毒力中的作用。方法采用长臂同源聚合酶链反应（LFH—PCR）的方法分别构建这4个序列的红霉素抗性基因（erm）替代缺失突变体,通过PCR和测序鉴定是否构建成功;通过绘制生长曲线观察基因对细菌生长繁殖的影响,通过小鼠毒力实验观察基因对细菌致病性的影响。结果所构建缺陷菌目的基因由eFm基因完全替代;spd—ABC、spd-1673和长间隔序列spd—J3个片段缺陷菌的生长趋势与野生菌没有明显差异,生长大约5hA值均可达到峰值,而spd-1672缺陷菌出现明显的延迟现象,生长8h后其4值才达到最高值;野生菌与缺失spd—ABC、spd-1673和长间隔序列spd—J的缺陷菌感染小鼠各组半数死亡时间分别为19、22、24和24h,差异无统计学意义,而spd-1672缺陷菌感染小鼠的半数死亡时间在75h左右,显著长于野生菌感染小鼠组（P〈0．05）。结论在构建的4个单个基因缺失突变体中,spd-1672缺陷后．S．pn生长明显延迟,毒力显著下降,提示spd-1672是s-Pn的一个新的毒力因子。     

The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.     

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.