Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology

Agarose-coated glass slides, after activation, were spotted with amine-modified oligonucleotide probes using a manual eight-pin arraying device. Two probes, designed to identify two common greenhouse fungal plant pathogens, Didymella bryoniae and Botrytis cinerea, were hybridized with polymerase chain reaction (PCR)-amplified fluorescently labeled DNA extracted from pure culture and from diseased plant tissue. The probes easily distinguished these pathogens from each other without cross reaction. Thickness of the agarose layer and length of the sample DNA were important factors affecting hybridization efficiency of immobilized probe to PCR product. These factors did not affect hybridization with short complementary oligonucleotide. Probes fixed on agarose-coated slides could differentiate samples as readily as probes on nylon but with potentially higher spot density and gave much better signal than probes on silylated slides. The use of plain glass slides, agarose, and a manual arrayer makes this technique useful for developing specialized and inexpensive DNA microarrays on a solid rigid substrate.     

Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty

Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.     

Avoidance of nonspecific hybridization by employing oligonucleotide micro-arrays generated from hydrolysis polymerase chain reaction probe sequences

We have developed a low-density oligonucleotide-based micro-array where 5'-end-tethered capture probe sequences were derived from Primer Express software. The capture probes represent hydrolysis probe sequences devoid of any fluorochromes and were shown to retain hybridization binding specificity to their amplicons; hybridization specificity was retained independent to probe sequences. This procedure allowed the specificity of each capture probe to be verified using real-time polymerase chain reaction (PCR) in the presence of nucleic acid sequences typically expected to be present within a sample and therefore has reduced possibility of nonspecific hybridization when used in a micro-array format. We propose that specificity-validated probes are applied to form a micro-array for the purpose of general target screening, with incumbent multiparallelization and cost and time savings. However, if required, the subset of probe sequences of interest can be used for quantitative assessment in conventional real-time PCR.     

Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays

Efficiencies of mismatch discrimination using size-varied capture probes were examined at various hybridization temperatures. The probes were 17, 15, 13, 11, 9, and 7 nucleotides long and contained single-base mismatches at their 3' ends. The optimal signal intensity and efficiency of base stacking hybridization on mismatch discrimination were observed for capture probes with a melting temperature (Tm) value of 36 degrees C, in the detection of DNA sequence variations at 40 degrees C. We employed asymmetric PCR to prepare single-stranded target DNA labeled with a fluorescent dye, and the PCR product was hybridized on the DNA microarray with no further purification. Our efforts have enhanced the sensitivity and simplified the procedures of base stacking hybridization on mismatch discrimination. As a model experiment, this improved technology was used to identify plasmid templates of human leukocyte antigen (HLA)-A alleles 2601, 2902, and 0206 on oligonucleotide microarrays. It is now possible to apply this simple, rapid, sensitive, and reliable base stacking hybridization technology to detect DNA sequence variations on microarrays in clinical diagnosis and other applications.     

Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection.

We have developed a simple gene quantification system using the competitive polymerase chain reaction (CPCR) followed by microtiter format analysis. CPCR is carried out using a mutant competitor with the same size as the target DNA product, and a minimal base exchange to insure the same amplification kinetics. One primer is aminated at the 5' end to produce PCR products that are captured onto carboxylated wells of microtiter plates through peptide bond formation. The non-aminated DNA strands are stripped off from the wells by alkali washing, and the remaining aminated strands are hybridized with either a digoxigenin-labeled wild type-specific oligonucleotide probe or a competitor-specific probe. To standardize the hybridization conditions of the probes, a DNA construct containing wild type and mutant competitor sequences in tandem is captured at different concentrations, hybridized with the probes, and used to generate a standard curve. Bound probes are detected by anti-digoxigenin antibody conjugated with peroxidase and chromogen. Optical densities are recorded with a conventional microtiter plate reader and converted to concentrations according to the standard curves. The ratios of wild type DNA to mutant competitor are used to determine the initial amounts of wild type DNA in the samples. This method was used successfully to quantify human immunodeficiency virus type 1 (HIV-1) env gene in human lymphocytes. It only requires a thermal cycler and a conventional microtiter plate reader, and can be readily done on a large scale. Potential applications include detection of other pathogens, diagnosis of genetic disorders and studies of gene expression.     

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.     

Tag/hybridization-based sensitive detection of polymerase chain reaction products

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

Detection and modelling of DNA hybridization by EIS measurements. Mention of a polythiophene matrix suitable for electrochemically controlled gene delivery

A conducting polymer sensor for direct label-free DNA detection based on a polythiophene bearing an electroactive linker group is investigated. DNA hybridization is studied by electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) techniques. Modelling of DNA hybridization by EIS measurements exhibits the contribution of nucleic acid to a superficial p-doping process. A 675-mer single-stranded DNA is produced using asymmetric PCR from a DNA sequence of a transposable element mariner and hybridized to the previously immobilized probe. Electrochemical stimulus leads to the release "on demand" of DNA fragments and the amount delivery permits to do PCR amplification.     

Effect of salicylic acid,methyl jasmonate,ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes

A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.     

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Purification of single stranded DNA from asymmetric PCR product using the SMART system

A method has been developed using the SMART system for the purification of single stranded DNA from a mixture containing single- and double-stranded DNA amplified using asymmetric PCR. The asymmetric PCR product was separated into single- and double-stranded DNA using an anion exchange column which took 15 min. Compared to another method in which biotinylated symmetric PCR products were applied to an immobilized streptavidin column, this method was simple and could purify single- and double-stranded DNA. © Rapid Science Ltd. 1998     

Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescent adsorbent.

We studied various parameters affecting the sensitivity of assays that use nucleic acid hybridization in solution followed by capture of the hybrid on a solid phase. Sensitivity is limited not only by nonspecific binding of the detection components but also by reannealing of the target or probe to itself. To perform sensitive assays, the probe concentration must be low enough to reduce high nonspecific binding. Under these conditions, however, the strand displacement reaction or the reannealing of the target to itself drastically decreases the hybridization yield, particularly when the target and the probes are different sizes. To improve DNA detection, we propose a sandwich method based on hybridization of oligonucleotides with a single-strand DNA obtained by polymerase chain reaction under asymmetric conditions. The assay can be performed in one step using a bioluminescent detection procedure which does not require any separation step. The specificity of the method is sufficient to perform a rapid detection and quantification of papillomavirus in biological samples.