Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

Enhancement of a hybridization analysis efficiency by the controlled DNA fragmentation

The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes.     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

Enhancement of hybridization analysis efficacy by means of controlled fragmentation of analyzed DNA

The influence of fragmentation of analyzed DNA amplicon on the efficacy of specific sequence detection by means of heterophase hybridization analysis was investigated. The detection of DNA sequence was carried out colorimetrically after introduction of biotin label into the oligonucleotide probe immobilized on a solid support upon its limited elongation in the complex with the analyzed DNA using Taq polymerase. Two simple and reproducible approaches to DNA analyte fragmentation were suggested. They are based on the formation apurinic/apyrimidinic sites in DNA followed by their degradation upon the thermal treatment. Apurinization of DNA was achieved with a mild acidic treatment. The apyrimidinic sites were formed when DNA fragment containing dTMP and dUMP residues in various ratios was treated with uracil-DNA-glycosylase (UDG). DNA analytes pretreated by one of these approaches can be used without additional purification for hybridization analysis of DNA with the use of Taq polymerase. The efficacy of hybridization analysis is shown to be higher in the case of the fragmented DNA in comparison with the native DNA amplicon. The use of fragmented DNA analytes allows utilizing bridged oligonucleotides as highly selective probes with reduced hybridization properties.     

Nuclear and mitochondrial DNA have sequence homology with a chloroplast gene

Summary A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P₇₀₀ chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.     

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.     

Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.     

DNA hybridization detection with organic thin film transistors: toward fast and disposable DNA microarray chips

We demonstrate a novel DNA hybridization detection method with organic thin film transistors. DNA molecules are immobilized directly on the surface of organic semiconductors, producing an unambiguous doping-induced threshold voltage shift upon hybridization. With these shifts, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are differentiated successfully. This method is expected to result in higher sensitivity than the main competitive technology, ISFET-based sensors because of the direct exposure of DNA molecules to sensitive layers. Factors that influence sensor sensitivity have been analyzed and optimum conditions have been determined using statistically designed experiments. Under the optimum conditions, the maximum difference between saturation current ratios caused by ssDNA and dsDNA reaches as high as 70%. In order to make DNA detection fast, we also demonstrate rapid on-chip electrically enhanced hybridization using the TFTs. These technologies together will enable the realization of disposable, rapid-turnaround tools for field-deployable genomic diagnosis.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5′-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.     

A rapid method of gene detection using DNA bound to Sephacryl

A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay. This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA. The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B). Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction. Hybridization in this instance will result in the resin being radioactively labelled. The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method. The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling. (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization. Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected. The method has been used to distinguish between DNA from sickle, heterozygote and normal patients.     

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.     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

Development of DNA diagnostic methods for the detection of new fish iridoviral diseases

A new disease of epidemic proportions caused by fish viruses within the Iridoviridae family inflicts serious damage on red sea breams (Pagrus major) and striped jack (Caranx delicatissimus) populations grown in aquacultures in Japan. A partial segment of the fish iridoviral DNA was directly amplified using the polymerase chain reaction (PCR) with synthetic primers designed from well conserved nucleotide sequences between the frog virus 3 (Ranavirus) and the silkworm iridescent virus type 6. The deduced amino acid sequence from the nucleotide sequence of the PCR fragment demonstrates a high correlation with a partial sequence from the frog virus 3. Using the PCR method with specific primers, we could detect three of four different known types of fish iridoviruses in diseased fishes. To construct more reliable detection methods specific for this viral family, DNA fragments which can specifically hybridize with all of the four known iridoviridae viral DNAs were screened from the genomic library of one iridoviridae strain. The hybridization assay, using a specific fragment which contains regions which are highly homologous with a characterized partial sequence from the frog virus 3, proved to be a reliable diagnostic tool for fish iridoviral diseases.     

Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS.

The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the melting temperature ( T (m)) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Colorimetric quantification of in vitro-amplified template DNA to be used for solid phase sequencing.

A protocol for colorimetric determination of DNA amplified by the polymerase chain reaction (PCR) and subsequently immobilized to a solid support is described. The protocol consists of three steps: (i) binding of PCR amplified lac operator-containing DNA to magnetic beads; (ii) binding of a Lac repressor-beta-galactosidase fusion protein to the lac operator and (iii) colorimetric detection of the immobilized beta-galactosidase. In practice, steps (i) and (ii) are performed concurrently. The protocol is well suited both for manual and automated procedures and the immobilized template can, after melting, be used directly for solid phase sequencing. The assay is used to demonstrate that template concentration is important for the quality of sequence data obtained from an automated DNA sequencer.     

DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.