A simple and ultrasensitive electrochemical DNA biosensor based on DNA concatamers

A simple, ultrasensitive and selective electrochemical DNA biosensor based on DNA concatamers is described, which can detect as low as 100 aM target DNA even in complex samples.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.     

电化学DNA生物传感器*

对特异DNA序列的检测在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的意义,DNA传感器的研究就是为了满足对特异DNA序列的快速、便捷、高灵敏度和高选择性检测的需要。近年来涌现出了多种传感策略,根据检测方法的不同可以大致分为光学传感器、电化学传感器、声学传感器等。由于电化学检测方法本身所具有的灵敏、快速、低成本和低能耗等特点,电化学DNA传感器已成为一个非常活跃的研究领域并在近几年中得到了快速发展。本文概括了近年来在DNA传感器的重要分支——电化学DNA传感器领域内的一些重要进展,主要包括DNA探针在传感界面上的固定方法和各种电化学DNA杂交信号的检测方法。     

Rapid assessment of DNA damage induced by polystyrene nanosphere suspension using a photoelectrochemical DNA sensor

Nanomaterials have been used increasingly in a wide variety of applications, and some of them have shown toxic effects on experimental animals and cells. In this study, a previously established photoelectrochemical DNA sensor was employed to rapidly detect DNA damage induced by polystyrene nanosphere (PSNS) suspensions. In the sensor, a double-stranded DNA film was assembled on a semiconductor electrode, and a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine...     

Molecular beacon probes for the detection of cisplatin-induced DNA damage

Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin.     

高灵敏纳米金比色芯片检测乙型肝炎病毒DNA及其变异

构建了新型纳米金比色芯片,利用Taq DNA连接酶的连接特异性,将其与乙型肝炎病毒DNA( HBV-DNA)靶序列完全互补杂交的捕获探针(固定在芯片上)和纳米金修饰的探针连接成一条链,从而将纳米金颗粒固定到芯片点阵上,再通过银染反应放大,形成裸眼可见的显色信息.通过点阵的位置及灰度,即可判断HBV-DNA靶序列的单碱基突变,并得出相对定量信息.本实验对不同浓度的HBV-DNA靶序列进行了检测.结果显示:此技术对单碱基突变有很强的特异性识别能力,并且具有较高的灵敏度(约10 pmol/L),在10～100 pmol/L浓度范围内表现出较好的线性关系.该技术检测时间短(＜1 h)、操作简单、不需要特殊的检测设备,具有很好的临床应用前景.     

The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence-specific DNA structure variations

Curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved sequences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence-specific DNA twist and wedge angles.     

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.     

寡聚酰胺为探针的电化学DNA生物传感器

DNA分子中的碱基对可以长程传递电荷, DNA分子中的碱基π堆积结构为电荷的长程传递提供了良好的通道. 电荷在DNA分子中的传递受碱基序列的影响, 利用这种性质可以构建DNA碱基错配检测的电化学传感器. 寡聚酰胺能和DNA以小沟绑定方式高亲和力地结合, 并且具有序列识别功能, 本文以带有硝基官能团的寡聚酰胺分子为电化学探针, 设计了电化学DNA生物传感器. 结果显示, 寡聚酰胺与DNA修饰电极作用后, 电化学响应显著增强, 并且可以作为检测DNA碱基错配的电化学探针分子.     

Detecting DNA methylation through changes in transverse proton relaxation

We present a facile, simple method to detect DNA methylation by measuring the transverse proton relaxation behaviour. Positively charged nanoparticles are arranged along the negatively charged backbone of DNA strands through electrostatic interactions. The arrangement of NPs along DNA strands aids to amplify and compare the transverse proton relaxation signal for un-cut versus cut DNA strands cleaved by sequence specific restriction enzymes. Results from this study suggest that the presence of methylation on DNA can be detected using superparamagnetic NPs using NMR.     

Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes

We herein report a novel nanoparticle-based electrochemical DNA detection approach. This DNA sensor is based on a "sandwich" detection strategy, which involves capture probe DNA immobilized on gold electrodes and reporter probe DNA labeled with gold nanoparticles that flank the target DNA sequence. Electrochemical signals are generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to surface-confined capture probe DNA via electrostatic interactions. We demonstrated that the incorporation of a gold nanoparticle in this sensor design significantly enhanced the sensitivity and the selectivity. Nanoscale control of the self-assembly process of DNA probes at gold electrodes further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar (zeptomoles) DNA targets and exhibited excellent selectivity against even a single-base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

A dual-amplification system for colorimetric DNA detection based on the assembly of biomolecules

A sensitive and selective colorimetric biosensor for the detection of DNA is described, which combines the amplification of self-assembly with the enrichment of horseradish peroxidase.     

Dielectrophoretic isolation of DNA and nanoparticles from blood

The ability to effectively detect disease-related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak-to-peak and 10 kHz, a wide range of high molecular weight (HMW)-DNA and nanoparticles were concentrated into high-field regions by positive DEP, while the blood cells were concentrated into the low-field regions by negative DEP. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected by fluorescence. HMW-DNA could be detected at 260 ng/mL, which is a detection level suitable for analysis of disease-related cell-free circulating DNA biomarkers. Fluorescent 40 nm nanoparticles could be detected at 9.5 × 10(9) particles/mL, which is a level suitable for monitoring drug delivery nanoparticles. The ability to rapidly isolate and detect DNA biomarkers and nanoparticles from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time and complexity.     

VISUALIZATION OF ULTRAVIOLET LIGHT-INDUCED THYMINE DIMERS IN DNA BY IMMUNOELECTRON MICROSCOPY

Abstract— To see the damage of DNA due to ultravoilet-B more distinctly, immunoelectron microscopic studies using a monoclonal antibody against cyclobutane-type thymine dimers were performed. As a result, we could detect the existence of thymine dimers on human genomic DNA and _pUC18 plasmid DNA visually. This technique can be useful to locate the photoproducts formed on DNA.     

Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

新型DNA电化学传感器的研制及其用于DNA氧化性损伤检测的研究

用溶胶-凝胶法在玻碳电极上制备了纳米多孔羟基磷灰石(HAp)-聚乙烯醇(PVA)涂层膜固定双链DNA,得到了一种新型DNA电化学传感器,检测了由Fenton反应引起的DNA氧化性损伤.结果表明,一定量浓度的抗坏血酸(AA)能加速Fenton反应的进行,使DNA损伤很快达到极限;损伤试剂中Fe²⁺的浓度越大,产生的羟基自由基(OH.)越多,对DNA的损伤就越严重;损伤试剂中EDTA的浓度越小,溶液中游离的Fe²⁺以及与DNA键合的Fe²⁺的浓度则相对越大,对DNA的损伤也就越严重.