Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed-side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel-based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.     

Determination of DNA by sub- and super-equivalence method of isotope dilution analysis using enzyme reaction

Sub- and super-equivalence method of isotope dilution analysis (SSE-IDA) using enzyme reaction was first applied for the determination of biological substance, DNA. Radioactive DNA (pUC18) to be analysed was prepared by incorporating 3H-thymidine in growing E. coli. A part of DNA was cut into the linear form (L-form) DNA under definite conditions using a restriction enzyme HindIII, following the separation of each by gel electrophoresis. Radioactivites of separated L-form DNA of two series were measured. The quantity of DNA was obtained by a graph method of SSE-IDA. As preliminary experiments, it was examined under what conditions the enzyme reaction proceeds as zeroth or first order reaction. We used the enzyme and substrate concentrations near zeroth order, where ordinary subst-IDA seems not to give a satisfactory results. As the results, 0.25 microgram of DNA was determined the error of about 10%.     

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.     

Determination of Cobalt(II)-Hydrogen Peroxide-Induced DNA Oxidative Damage and Preventive Antioxidant Activity by CUPRAC Colorimetry

Abstract

The classical Fenton system composed of Fe(II) and H₂O₂ uses harsh oxidative conditions and cannot realistically simulate physiological oxidations which are less severe. Here, reactive oxygen species (ROS) were generated with a combination of CoSO₄ and H₂O₂ to provide milder conditions. DNA was used as a biologically meaningful probe for monitoring the oxidative conversion. Oxidative hazard on DNA was accomplished in ammonia/ammonium chloride buffer at 37?°C, and the Fenton reaction was stopped with trichloroacetic acid (TCA). A suitable aliquot of this solution was added to cupric ion reducing antioxidant capacity (CUPRAC) reaction mixture, and the absorbance at 450?nm was recorded. The oxidized species derived from DNA were CUPRAC-reactive while intact DNA was not. The protective effects of antioxidants (AOxs), known to have radical scavenging effects, were tested; green tea and a synthetic fetal bovine serum (FBS) were also successfully used as real ROS scavengers. Although the classical iron-based Fenton procedure applied in ethanol medium generated CUPRAC-responsive products, the proposed system was perfectly ethanol-tolerant, enabling the CUPRAC measurement of DNA oxidation products against an unaffected reagent blank. The protective effects of phenolic antioxidants, perfectly solubilized in ethanol, could also be measured.     

Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood.

A high-performance capillary electrophoresis system with a polysiloxane-coated capillary and polymeric buffer additives was investigated for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. Mobility data and Ferguson plots of the DNA fragments at different polymer (hydroxypropylmethylcellulose) concentrations indicated that effective molecular sieving was obtained consistent with existing data of conventional gel electrophoresis and with recent HPCE data. The precision and peak efficiency were excellent and the system was applied to the analysis of specific co-amplified DNA sequences (HIV-1 and HLA-DQ-alpha). After PCR, ultrafiltration was used in the sample preparation step to desalt the sample and to remove superfluous PCR reaction products. Electrokinetic injection was used for sample introduction into the capillary. The addition of ethidium bromide to the buffer resulted in longer migration times of DNA fragments and better peak resolution. During HPCE, an artifact associated with dilute DNA solutions leading to the appearance of extra peaks in the electropherogram was found.     

Determination of nucleic acids based on shifting the association equilibrium between tetrasulfonated aluminium phthalocyanine and acridine orange

Based on the ability of nucleic acids to shift the association equilibrium of the ion-association complex of Acridine Orange and tetrasulfonated aluminium phthalocyanine, thus leading to an increase in the phthalocyanine fluorescence, a method is suggested for the fluorimetric determination of nucleic acids. Investigations were carried out on the spectral characteristics, order of addition of reagents, selection of the buffer system, effect of pH, influence of reaction time, effect of salt, the usage of reagents, interference of foreign substances and the effect of different acridine derivatives. Under the optimum conditions, the calibration graphs for the determination of calf thymus DNA (CT DNA), salmon DNA (SM DNA) and yeast RNA were linear over the ranges 0.04-1.2, 0.04-1.2 and 0.1-1.2 micrograms cm-1, respectively. The detection limits for CT DNA, SM DNA and RNA were 17, 24 and 98 micrograms cm-3, respectively. The relative standard deviation (n = 6) was within 4.6% for the detection of samples. The method was applied to the determination of Staphylococcus aureus DNA and the result was in agreement with that achieved by a UV method.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

Synthesis of mercaptoethylamine-coated CdSe/CdS nanocrystals and their use for DNA probe.

CdSe/CdS nanocrystals (NCs) have been synthesized in aqueous solution by using mercaptoethylamine as a stabilizer. The results of TEM, UV-Vis and FL spectra show that the product is of excellent crystal structure, uniform in radius, with extraordinary fluorescence characters. These CdSe/CdS NCs allow the ultrasensitive quantitative detection of DNA. Under the optimum conditions, linear relationships have been found between the relative fluorescence intensity and the DNA concentration in the ranges 0 - 10 and 10 - 100 microg mL(-1); the linear equations are DeltaF = 210 + 80.91 C and DeltaF = 946 + 10.57 C (C, microg mL(-1)), respectively. The detection limits are 0.251 and 1.920 microg mL(-1), correspondingly. The proposed method has been applied to the determination of DNA in pig liver. It is indicated that these NCs could become a newly kind of DNA probe. In addition, the mechanism of the binding reaction has also been explored. It is considered that the binding reaction is mainly due to static electricity forces.     

Rational Engineering of a Dynamic,Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy‐driven DNA nanomachine with three‐dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy‐driven catalytic reaction of intramolecular toehold‐mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye‐labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction‐based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity‐induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

Facile synthesis of 2,3,5,6-tetrabromo-4-methyl-nitrocyclohexa-2,5-dien-1-one, a mild nitration reagent

Nitrocylcohexadienones have been applied as nitration reagents for mild, mono-nitrating reactions. The original synthesis of 2,3,5,6-tetrabromo-4-methyl-4-nitrocylcohexa-2,5-dien-1-one appeared to be difficult to pursue due to both the solvent system and reaction conditions. Therefore, we applied a modified solvent system and optimized the reaction conditions to prepare the dienone at 0°C, eventually overcome the difficulties.     

DNA‐Mediated Proximity‐Based Assembly Circuit for Actuation of Biochemical Reactions

Smart nanodevices that integrate molecular recognition and signal production hold great promise for the point‐of‐care (POC) diagnostic applications. Herein, the development of a DNA‐mediated proximity assembly of biochemical reactions, which was capable of sensing various bio‐targets and reporting easy‐to‐read signals is reported. The circuit was composed of a DNA hairpin‐locked catalytic cofactor with inhibited activity. Specific molecular inputs can trigger a conformational switch of the DNA locks through the mechanisms of toehold displacement and aptamer switching, exposing an active cofactor. The subsequent assembly of an enzyme/cofactor pair actuated a reaction to produce colorimetric or fluorescence signals for detecting target molecules. The developed system could be potentially applied to smart biosensing in molecular diagnostics and POC tests.     

Rational Engineering of a Dynamic,Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy-driven DNA nanomachine with three-dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy-driven catalytic reaction of intramolecular toehold-mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye-labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction-based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity-induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

A study on photolinkers used for biomolecule attachment to polymer surfaces

The use of photolinkers (photoactivatable heterobifunctional crosslinkers) is a popular method to attach biomolecules to polymer surfaces. This study addresses the selection of photolinker and the adjustment of reaction conditions, such as the concentration of biomolecule applied, and irradiation time. The influence of these variables are investigated for four prominent photolinkers: ketyl-reactive benzophenone (BP) and anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD). The influence of substrate material is discussed, and three different polymers served as representative substrates: poly(methyl methacrylate) (PMMA), polystyrene (PS), and a cycloolefin copolymer (COC). We compared the overall photolinking efficiency of all photolinkers with respect to the polymer substrate they are applied to, and we found considerable differences for certain photolinker/substrate combinations. Of all photolinkers and substrates tested, PTD as photolinker and COC as substrate showed the highest photolinking efficiencies and fastest reaction times. For this study DNA oligonucleotides were chosen as a model system of biomolecular probes, and fluorescence detection of DNA microarrays served as method of detection.     

Mild and efficient cyclization reaction of 2-ethynylaniline derivatives to indoles in aqueous medium

Results of the optimized cyclization reaction of 2-ethynylaniline derivatives to indoles catalyzed by copper(II) salts are described. The reactions can be carried out in a mixture of H₂O and MeOH in the presence of 1-ethylpiperidine at room temperature. These conditions can be applied to a bulky substrate, which is difficult to be cyclized efficiently by existing reaction conditions. Furthermore, this reaction condition was applied to a catalyst recycling reaction system.     

吖啶橙(AO)-藏红T(ST)荧光探针测定DNA的研究

简述了以荧光能量转移本系测定ＤＮＡ方法的发展过程，同时对吖啶（ＡＯ）－藏红Ｔ（ＳＴ）荧光探针测定ＤＮＡ的条件进行分析，证明了该法灵敏度高、线性范围宽、检出限低、选择性好、抗干扰能力强，该法为微曦ＤＮＡ测定分析工作提供了一个新的途径。     

DNA对Ru(bpy)_2(dppx)~(2+)-SDS体系共振光散射的猝灭作用及其应用

研究了Ru(bpy)2(dppx)2+-SDS-DNA(bpy=2,2′-联吡啶,dppx=7,8-二甲基-吡啶并[3,2-a:2′,3′-c]吩嗪)体系的共振光散射光谱。结果表明,在阴离子表面活性剂十二烷基硫酸钠(SDS)预胶束聚集体存在下,Ru(bpy)2(dppx)2+-SDS体系具有很强的共振光散射,DNA的加入使其共振散射光猝灭。探讨了反应机理。基于DNA对Ru(bpy)2(dppx)2+-SDS体系共振光散射的猝灭作用,建立了共振光散射法测定DNA的新方法。在最佳实验条件下,体系在393nm处的共振光散射猝灭程度与DNA的浓度呈线性关系,线性范围为0.01～1.2mg/L,检出限为1.5μg/L。     

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 CT).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.     

A novel and sensitive method for recognition and indirect determination of Al(III) in biological fluid based on the quenching of resonance Rayleigh scattering intensities of "Al(III)-EV-DNA" complexing system

A novel method for recognition and indirect determination of Al(III) by using biological molecules has been established based on the quenching of RRS intensity. In the weak acidic medium, the reaction of ethyl violet (EV) and DNA would result in great enhancement of RRS intensity. However, the presence of Al(III) would lead to the decrease of the RRS intensity owing to the competition coordination of Al with DNA. The decreased intensity of RRS is directly proportional to the concentration of Al(III) in the range of (0.1-2.5)x10(-6) and (0.30-4.5)x10(-5)M, respectively. The method has high sensitivity and its detection limit (3sigma) is 3.6x10(-8)M. The characteristics of RRS spectra of the system, the optimum conditions of the reaction, and the reaction mechanism have been investigated. The method can recognize Al(III) selectively owing to its strong binding to the phosphate backbone of DNA, and has been applied to the determination of Al(III) concentration in synthetic biological samples with satisfactory results. Therefore, the proposed method is promising as an effective means for selective recognition and sensitive determination in situ of Al(III). Furthermore, this study would contribute to further understanding of the biological significance of Al neurotoxicity.     

Hyperbranched Hybridization Chain Reaction for Triggered Signal Amplification and Concatenated Logic Circuits

A hyper‐branched hybridization chain reaction (HB‐HCR) is presented herein, which consists of only six species that can metastably coexist until the introduction of an initiator DNA to trigger a cascade of hybridization events, leading to the self‐sustained assembly of hyper‐branched and nicked double‐stranded DNA structures. The system can readily achieve ultrasensitive detection of target DNA. Moreover, the HB‐HCR principle is successfully applied to construct three‐input concatenated logic circuits with excellent specificity and extended to design a security‐mimicking keypad lock system. Significantly, the HB‐HCR‐based keypad lock can alarm immediately if the “password” is incorrect. Overall, the proposed HB‐HCR with high amplification efficiency is simple, homogeneous, fast, robust, and low‐cost, and holds great promise in the development of biosensing, in the programmable assembly of DNA architectures, and in molecular logic operations.     

A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases.

BACKGROUND: Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. RESULTS: Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities. CONCLUSIONS: The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.