DNA Hybridization Reaction Research Articles

Detection of DNA Hybridization Properties Using Thermodynamic Method

The determination of DNA hybridization reaction can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and application area. So, the improvement of DNA hybridization detection method is very important for the determination of hybridization reaction. Several molecular biological techniques require accurate predictions of match versus mismatch hybridization thermodynamics, such as polymerase chain reaction (PCR), sequencing by hybridization, gene diagnostics and antisense DNA probes. In addition, recent developments of DNA chip array as means for biochemical assays and DNA sequencing requires accurate knowledge of hybridization thermodynamics and population ratios at match and mismatch target sites. In this study, we report the properties of the probe oligonucleotide and match, mismatch target oligonucleotide hybridization reaction using thermodynamic method. Thermodynamics of 5'-oligonucleotides with central and terminal mismatch sequences are obtained by measuring UV-absorbance as a function of temperature. The data show that the nearest-neighbor base-pair model is adequate for predicting thermodynamics of oligonucleotides with average deviations for enthalpy (ΔH0), entropy (ΔS0), free energy change at 37°C (ΔG370) and melting temperature (Tm), respectively.

Detection of DNA Point Mutation by Atom Transfer Radical Polymerization

We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.

Using a microfluidic device for 1 l DNA microarray hybridization in 500 s

This work describes a novel and simple modification of the current microarray format. It reduces the sample/reagent volume to 1 μl and the hybridization time to 500 s. Both 20mer and 80mer oligonucleotide probes and singly labeled 20mer and 80mer targets, representative of the T-cell acute lymphocytic leukemia 1 (TAL1) gene, have been used to elucidate the performance of this hybridization approach. In this format, called shuttle hybridization, a conventional flat glass DNA microarray is integrated with a PMMA microfluidic chip to reduce the sample and reagent consumption to 1/100 of that associated with the conventional format. A serpentine microtrench is designed and fabricated on a PMMA chip using a widely available CO2 laser scriber. The trench spacing is compatible with the inter-spot distance in standard microarrays. The microtrench chip and microarray chip are easily aligned and assembled manually so that the microarray is integrated with a microfluidic channel. Discrete sample plugs are employed in the microchannel for hybridization. Flowing through the microchannel with alternating depths and widths scrambles continuous sample plug into discrete short plugs. These plugs are shuttled back and forth along the channel, sweeping over microarray probes while re-circulation mixing occurs inside the plugs. Integrating the microarrays into the microfluidic channel reduces the DNA–DNA hybridization time from 18 h to 500 s. Additionally, the enhancement of DNA hybridization reaction by the microfluidic device is investigated by determining the coefficient of variation (CV), the growth rate of the hybridization signal and the ability to discriminate single-base mismatch. Detection limit of 19 amol was obtained for shuttle hybridization. A 1 μl target was used to hybridize with an array that can hold 5000 probes.

Amino-functionalized plasma polymer films for DNA immobilization and hybridization

DNA attachment on amine functionalized surfaces has been found to depend on the macromolecular architecture of the plasma films, on the amine group density as well as on the immobilization conditions. Surface Plasmon Resonance spectroscopy (SPR) and Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) were used to monitor (a) the immobilization of probe oligomers (25 mers) to plasma polymerised allylamine films (ppA) and (b) their subsequent hybridization reactions with fluorophore-labeled 15 mer target oligonucleotides from solution. To avoid unspecific adsorption of target DNA, any unreacted amino groups were neutralized with succinic anhydride according to standard hybridization procedures. SPF spectroscopic investigations allowed for a precise differentiation between hybridization reactions with mismatch 2 (MM2), mismatch 1 (MM1) and fully complementary (MM0) oligonucleotide target sequences. Complementary target DNA hybridization was shown to have a significantly higher affinity constant (KA) than MM1 and MM2. Low energy plasma deposited allylamine films appear to act as a 3D matrix showing a high capacity towards DNA immobilization and hybridization reactions.

Dynamic Microcantilever Sensors for Discerning Biomolecular Interactions

Response of a conductive micromechanical cantilever placed in close proximity to a surface undergoing electrical excitation near the resonance frequency of the cantilever is influenced by the presence of microscopic dielectrics in the gap between the cantilever and the sample surface. The variations of the resonance response of unmodified cantilevers at gap distances below a few hundred nanometers are used to discern biomolecular differences of oligomeric nucleic acids in an array format without the use of extrinsic labels. The resonance response variation paves the way for the development of high throughput detection of biomolecular reactions, such as DNA hybridization reactions or antibody-antigen interactions without the use of external labels, in which the need is only to see the presence or absence of interaction. This dynamic method is simple, does not require immobilizing individual elements on a cantilever array, and is compatible with current generation DNA chips in which DNA spots are deposited in micro- and nanoarray format.

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Pepper golden mosaic virus Affecting Tomato Crops in the Baja California Peninsula, Mexico.

In the state of Baja California Sur, tomatoes (Lycopersicon esculentum Mill.) are cultivated on approximately 1,000 ha. Occurrence of viral diseases is currently causing low yields and severe losses. Virus-like symptoms (severe leaf curling, stunting, reduced leaf size, and mosaic patterns) were observed on 99% of tomato plants in 2002 in La Paz, Baja California Sur. Whiteflies (Bemisia tabaci Gennadius) were present in affected fields and appeared to be associated with the disease. The virus was experimentally transmitted from infected plants to tomato and peppers seedlings by whiteflies and as infectious DNA (replicative form) by mechanical and biolistic inoculation. Symptoms similar to those found in the field were observed in experimental transmission assays. DNA from inoculated plants was extracted and analyzed by DNA hybridization and polymerase chain reaction (PCR) using degenerate (1) and specific (2) primers. The PCR products (1.1 kb) obtained from the common region (GenBank Accession No. AY368336) suggested the presence of a bipartite geminivirus. The nucleotide sequence of the PCR products showed a 98% identity to Pepper golden mosaic virus-Tamaulipas strain (PepGMV-Tam) in the intergenic region (IR). Similar identities (97%) were obtained by using the predicted amino acid sequences of the amino termini of the coat proteins. Identities in the replication proteins (92%) and IR iterative sequence analyses show that the PepGMV-La Paz isolate is a closely related strain of PepGMV-Tam. To our knowledge, this is the first report of PepGMV affecting tomato crops in Baja California Sur, Mexico.

Characterization of Pulsed Plasma Polymerization Allylamine as an Adhesion Layer for DNA Adsorption/Hybridization

This paper presents an investigation of allylamine polymerization in pulsed radio frequency (RF) plasma as an adhesion layer immobilizing DNA probe for DNA hybridization assay. We looked for a simple and innovative way to improve the stability of pulsed plasma polymerization allylamine (PPPAA) film in phosphate-buffered saline (PBS) solution. To better understand the mechanism of PPPAA stability and the influence on DNA adsorption from films, several techniques were used. Fourier transform infrared (FTIR), atomic force microscopy (AFM), surface contact angle, and surface plasmon resonance (SPR) as well as surface plasmon enhanced fluorescence spectroscopy (SPFS) were all employed to investigate the effect of plasma conditions on the film structure, amine density, and the DNA hybridization reaction. It concludes that polymer deposited at low working pressure is very stable and can be used as adhesion layers for further study of DNA hybridization assay.

A combined oligonucleotide and protein microarray for the codetection of nucleic acids and antibodies associated with human immunodeficiency virus, hepatitis B virus, and hepatitis C virus infections

A multiplexed assay based on the codetection of nucleic acids and antibodies in human serum infected by human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus was proposed. The combined immuno- and oligosorbent array (CombOLISA) microarray is prepared in 96-well standard microplates by spotting (1) nucleic probes specific for a virus genome, (2) viral proteins for the capture of serum antibodies, and (3) nonspecific proteins for verifying specificity. Experimental assay conditions were optimized so that both DNA hybridization and immunological reactions can be achieved simultaneously in the same well and buffer and all at the same temperature. A generic detection system based on the precipitation of an insoluble colorimetric substrate in the presence of enzyme-labeled antibodies or streptavidin was proposed. The optical density of each spot was correlated to the corresponding analyte concentration. The influence of critical parameters on CombOLISA performance such as serum concentration was studied. Calibration curves and sensitivity thresholds were established for each parameter. Serial dilutions of serum were correlated to results obtained with validated immunoassay platforms such as a microplate enzyme-linked immunosorbent assay or the VIDAS automat. Also, several HIV- and HBV-infected serum samples were tested independently by CombOLISA and VIDAS. Coefficients of variation for genomic and proteomic parameters vs spot density were below 15%.

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.

Flexoelectric origin of nanomechanic deflection in DNA-microcantilever system

The membrane theory is used to study the recently observed nanomechanical bending of cantilevers, which have processed biomolecular adsorption or biochemical reactions. To be different from entropy-controlling bending mechanism discussed before, we propose that the flexoelectric effect induces cantilever bending. With the introduction of flexoelectric spontaneous curvature, the relation between the bending and biopolymer character is constructed by a simple analytical formula. The cantilever motion induced by adsorption of single-strand DNA and DNA hybridization reaction is quantified analytically and our results show good agreement with experiments.

Frequency and Seasonal Distribution of Pear Psylla Infected with the Pear Decline Phytoplasma in California Pear Orchards

ABSTRACT Pear decline (PD) is an important disease of Pyrus communis fruiting cultivars in Europe, Asia, and the Americas. PD is caused by a phloem-limited phytoplasma that, in California, is transmitted from diseased to healthy trees by pear psylla, Cacopsylla pyricola. The percentage of phytoplasma-infected pear psylla has never been assessed in the United States in field-collected insects. Pear psylla were collected monthly from PD-infected trees from three orchards in northern California. Individual psylla were tested for the presence of PD phytoplasma, using both a quantitative DNA hybridization and PD phytoplasma-specific polymerase chain reaction (PCR) assays. The percentage of infected psylla ranged from 0 to 45% depending on the orchard, the month and year collected, and the method of detection. The PD phytoplasma was detected in both the winterform and summerform pear psylla. Significantly more infected psylla were detected with PCR than with DNA hybridization analysis in two of the three orchards. The number of PD phytoplasma per pear psylla was estimated to range from 1 x 10(6) to 8.2 x 10(7). The percentage of PD-infected pear psylla found in the three northern California pear orchards suggests that both winterform and summerform pear psylla could be important in the transmission of PD.

Identification of Phytoplasma Taxa and Insect Vectors of Peach Yellow Leaf Roll Disease in California.

Two peach diseases in California, western X-disease (WX) and peach yellow leaf roll (PYLR) are caused by two genetically distinct phytoplasmas. Based on symptoms alone, WX and PYLR cannot be reliably distinguished. The objectives of this study were to determine which phytoplasma was causing PYLR disease in peach orchards planted near pear orchards and which phytoplasmas were present in potential insect vectors captured in both peach and pear orchards. Leafhoppers and pear psylla were collected from yellow sticky traps placed in three pear orchards and four peach orchards located in the Sacramento Valley from 1994 to 1996. DNA was extracted from potential vectors and suspect diseased trees, and analyzed for the presence of the WX and PYLR phytoplasmas using strain-specific DNA hybridization or polymerase chain reaction (PCR) assays. The most abundant phytoplasma detected in diseased peach trees was the PYLR phytoplasma. In 1994 and 1995, 10 to 25% of groups of pear psylla tested positive for the PYLR phytoplasma, while no psylla groups tested positive for the WX phytoplasma. Only one captured leafhopper tested positive for the WX phytoplasma. These results indicate the pear psylla is the primary vector of PYLR in northern California.

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes.

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.

A Single DNA Molecule Nanomotor

Nanomotors consisting of single protein molecules are abundant in living systems. Here we report a nanomotor made of a single DNA molecule. The DNA nanomotor can adopt two distinct conformations, intramolecular tetraplex and intermolecular duplex. The nanomotor switches between the two conformations through alternating DNA hybridization and strand exchange reactions, which enables the nanomotor to perform an inchworm like extending−shrinking motion. When the single molecule nanomotor is loaded with two organic molecules, a fluorophore and a quencher, the motion can be viewed in real time by monitoring the fluorescent signal. The DNA nanomotor functions efficiently both in solution and on nanoparticle surfaces. Its simple yet stable structure, convenient operation, and high efficiency may make the DNA nanomotor practically useful for powering nanosystems in future applications.

Speciation and distribution of the members of the Anopheles punctulatus (Diptera: Culicidae) group in Papua New Guinea.

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An. farauti 2, An. koliensis Owen, and An. punctulatus Dönitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

Development and Characterization of DNA Hybridization Reaction on PDMS Microchip

Development and Characterization of DNA Hybridization Reaction on PDMS Microchip

Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling.

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.

A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.

Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.