Oriented and vectorial immobilization of linear M13 dsDNA between interdigitated electrodes--towards single molecule DNA nanostructures

The ability to control molecules at a resolution well below that offered by photolithography has gained much interest recently. DNA is a promising candidate for this task since it offers excellent specificity in base-pairing combined with addressability at the nanometer scale. New applications in biosensing, e.g. interaction analysis at the single molecule level, or nanobiotechnology, e.g. ultradense DNA microarrays, have been devised that rely on stretched DNA bridges. The basic technology required is the ability to deposit spatially defined, stretched DNA-bridges between anchoring structures on surfaces. In this paper we present two techniques for spanning 2 microm long dsDNA bridges between neighboring interdigitated electrodes (IDEs). The extended DNA used was linearized M13 dsDNA (M13mp18 7231 bp, ca. 2.5 microm length), either unmodified, or with chemical modifications at both ends. The first approach is based on the dielectrophoretic (DEP) concentration and alignment of linearized wild-type dsDNA. IDEs with 1.7 microm spacing are driven with an AC voltage around 1 MHz leading to field strengths in the order of 1 MV m(-1). The dsDNA is polarized and linearized by the force field and accumulates in the gap between two neighboring electrodes. This process is reversible and was visualized by fluorescence staining of M13 DNA using PicoGreen, as intercalating dye. The resulting dsDNA bridges and their orientation are discernible under the fluorescence microscope using fluorescent particles of different color. The particles are tagged with sequence specific peptide nucleic acid (PNA) probes that bind to the DNA double strand at specific sites. The second approach is based on asymmetric electrochemical modification of a gold IDE with 2.0 microm spacings followed by spontaneous or stimulated deposition of a chemically modified M13-DNA. One side of the IDE was selectively coated with streptavidin by electropolymerization of a novel hydrophilic conductive polymer in the presence of the binding protein. The second side was modified with gold nanoparticles by reductive plating from aqueous gold chloride solution. An asymmetric double stranded (ds) M13 DNA carrying a 5'-thiol group at one end and a 5'-biotin at the other end was obtained by polymerase chain reaction (PCR) using two differently labeled primers. For DNA bridges to form spontaneously the modified IDE was incubated over night with a 50 nM solution of the modified M13 DNA. Potential applications of DNA-bridge formation in biosensing and biotechnology are discussed.     

End-joining long nucleic acid polymers

Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two-step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 ± 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated examples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionanotechnology and in single-molecule experiments.     

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.     

Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.     

DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.     

Polymerizing immobilization of acrylamide-modified nucleic acids and its application

The immobilization of nucleic acids on solid supports has been widely used in the detection of DNA and other biomolecules in sensor technology. Because three dimensional (3-D) hydrogel matrixes offer significant advantages for capturing probes over more conventional two dimensional (2-D) rigid substrates and the ability to provide a solution-mimicking environment, they are becoming increasingly attractive as desired supports for bio-analysis. Acrylamide-modified nucleic acids and acrylamide monomers being polymerized directly to immobilize nucleic acids is only one-step chemical process which is not interfered by exterior surroundings, and the 3-D polyacrylamide gel fabricated by this method is not required to be activated by some labile chemical treatments. Moreover, the attachment is extremely stable to withstand the cycling process involved in the polymerase chain reaction (PCR). In this paper, the development of polymerizing immobilization of acrylamide-modified nucleic acids is reviewed, and its applications in DNA sequence high-throughput analysis including mutation analysis and the whole genome sequencing are summarized.     

Electrochemical study of the XNA on Gold microarray

A novel electrode array was developed based on the XNA on Gold trade mark microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold electrode. The electrochemical reaction of K4Fe(CN)6 was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)6 to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis.     

Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.     

Silanized nucleic acids: a general platform for DNA immobilization

We have developed a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products on unmodified glass surfaces. By covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solutions we have generated a new class of modified nucleic acids, namely silanized nucleic acids. Such silanized molecules can be immobilized instantly onto glass surfaces after manual or automated deposition. This method provides a simple and rapid, yet very efficient, solution to the immobilization of prefabricated oligonucleotides and DNA for chip production.     

A rapid assay for affinity and kinetics of molecular interactions with nucleic acids

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.     

Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction

A method for the rapid preparation of DNA is described. The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system. A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs. Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region. Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample. The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion. This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins. The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents. With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase. The paper describes a method for DNA isolation that is rapid, simple and economical.     

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.     

Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

Surface plasmon field-enhanced fluorescence spectroscopy was employed to extensively investigate the hybridization behaviors of polymerase chain reaction (PCR) amplicons on a peptide nucleic acid (PNA) or DNA probe layer that was previously attached on a streptavidin-modified gold surface via biotin/streptavidin interaction. Despite the neutral backbone of PNA, the hybridization reactions were strongly influenced by the variation of ionic strength. The association rates exhibited a monotonic decrease with ionic strength increase and the maximum hybridization signal was achieved at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA/DNA electrostatic barrier, particularly in lower ionic strength range (e.g., 10 approximately 150 mM Na(+)). The electrostatic cross talk was shown to be largely reduced if the PNA probe layer was sufficiently diluted by following a strategic templated immobilization method. As a consequence, a pseudo-first-order kinetic model was applicable to describe the hybridization kinetics, and affinity constants were derived for evaluating the influence of single nucleotide polymorphisms (SNPs).     

Ultrasensitive electrical detection of nucleic acids by hematin catalysed silver nanoparticle formation in sub-microgapped biosensors

An ultrasensitive electrical detection method of nucleic acids has been demonstrated on sub-microgapped biosensor. In this method, peptide nucleic acid (PNA) probes were firstly immobilized in the gap areas of a pair of interdigited microelectrodes and then were hybridized with their complementary target DNA. After hybridization, hematin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hematin molecules act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Nearly 1fM sensitivity was achieved under optimal conditions. This approach is also applicable to the detection of RNA.     

Retention of enzyme by electropolymerized film: a new approach in developing a hypoxanthine biosensor

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para-diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme-based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5-300 muM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4 degrees C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.     

A fluorimetric method for the determination of nucleic acids using Ce(IV) and sodium triphosphate.

A novel and stable fluorimetric method was established for the determination of nucleic acids. The proposed method is based on the reduction by nucleic acids of Ce(IV) to fluorescent Ce(III). The fluorescence intensity can be greatly increased by sodium triphosphate. The enhanced fluorescence intensity is proportional to the concentration of nucleic acids in the range 4.2 x 10(-8)-4.2 x 10(-6) g/mL for fish sperm DNA and 5.0 x 10(-8)-6.5 x 10(-6) g/mL for yeast RNA, and the detection limits (S/N = 3) are 13.5 ng/mL and 45 ng/mL, respectively. The reaction mechanism of the hydrolytic scission of nucleic acids by Ce(IV) is discussed.     

SNP与基因检测及表达中的RCA技术

滚环扩增(rollingcircleamplification,RCA)技术是一种新的分子生物学检测方法。该方法不仅可以在体外等温条件下对核酸进行高度特异性的检测,而且还可通过线性或指数扩增来进行信号级联放大,其灵敏度能达到1个拷贝的核酸分子,因此,可用于痕量分子的检测。目前,滚环扩增技术广泛应用于全基因组DNA检测、核酸测序、单核苷酸多态性、DNA芯片及蛋白质芯片分析等领域。     

Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids.     

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic beta-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin-biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.     

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 A of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)-PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile-PP layers. The oligonucleotide arrays fabricated on HMDS-PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.