Generic Oligonucleotide Research Articles

Virus de la hoja rizada de calabaza (SLCV): diagnóstico, dinámica poblacional del vector y distribución espacio-temporal del virus

Los miembros del género begomovirus se transmiten por Bemisia tabaci y causan severas pérdidas en las cucurbitáceas. Los objetivos del estudio fueron: i) investigar la etiología de la enfermedad provocada por el virus de la hoja rizada de la calabaza; ii) conocer la dinámica de población del vector, comparar la efectividad de trampas amarillas pegajosas contra charolas amarillas y determinar el efecto del sitio de ubicación de las trampas, en el monitoreo de B. tabaci durante el ciclo de desarrollo del cultivo; y iii) estudiar la distribución espacio-temporal y severidad de virosis en calabaza. El estudio se realizó en la estación experimental del Colegio Superior Agropecuario del estado de Guerrero, ubicado en el Valle de Cocula. Se realizó la extracción del ADN a partir de hojas de calabaza (Cucurbita pepo) var. Grey Zucchini con síntomas de virosis. El PCR se llevó a cabo en cinco muestras con los oligonucleótidos genéricos para begomovirus, se secuenciaron dos productos de PCR y se generó un árbol filogenético. Se realizó la prueba de transmisión del virus en calabaza, en condiciones controladas. La dinámica poblacional de B. tabaci se determinó utilizando trampas amarillas pegajosas y charolas amarillas. A partir, de la sintomatología, se obtuvo el porcentaje de severidad y el área bajo la curva del progreso de la enfermedad (ABCPE). El análisis de PCR, secuenciación y filogenia, confirmaron la presencia del virus SLCV (KX620945.1-DAAV-1) en calabaza. Se comprobó que B. tabaci transmite a SLCV que reduce el crecimiento de la planta. La trampa pegajosa y fecha de muestreo influyen en la fluctuación poblacional de B. tabaci. La incidencia de B. tabaci y severidad de SLCV se manifestaron en todas las etapas fenológicas del cultivo y se relacionaron con el nivel de temperatura.

A bridge-like solution for Universal Microarray applications

In this work, we provided the evidence of an alternative DNA detection methodology, in the DNA microarray domain, based on a Universal Microarray approach. Despite the classic hybridization of an oligonucleotide immobilized onto a proper surface with a DNA target extracted from a biological sample and amplified by a PCR reaction, here we reported an alternative method based on the usage of generic oligonucleotides microarray sequences, coupled to special probes contained in the hybridization buffer, at end user level. These special probes present one region complementary to the oligonucleotide on the surface (microarray) and the other part recognizing a certain DNA target sequence. These special probes act like a bridge connection and the final hybridization leads to a three-units complex formation. We demonstrated that the hybridization occurs, and therefore the DNA target recognition, although with less efficiency, that is reasonable because of the thermodynamics constrains in the formation of the unstable three-units complex. However, signal detection is still comparable with standard hybridization.

Labeling Deoxyadenosine for the Preparation of Functional Conjugated Oligonucleotides

Herein we present a versatile synthetic method for the 8-thioalkylation of (deoxy)adenosine with a short carbon linker having on the other side a variety of molecules (psoralen, acridine) and functional groups (alkyne). After conventional protections, the modified adenosine can be phosphytylated and inserted into an oligonucleotide without affecting the standard protocols for supported oligonucleotide synthesis. The hybridization properties of a generic oligonucleotide containing the above conjugated moieties toward both DNA and RNA are evaluated both in the case of a perfectly complementary strand and in the case of a single mismatch. This methodology is suitable for the preparation of several types of derivatives and—through the alkynyl moiety—provides fast access to click-chemistry transformations.

Training a molecular automaton to play a game

Research at the interface between chemistry and cybernetics has led to reports of 'programmable molecules', but what does it mean to say 'we programmed a set of solution-phase molecules to do X'? A survey of recently implemented solution-phase circuitry indicates that this statement could be replaced with 'we pre-mixed a set of molecules to do X and functional subsets of X'. These hard-wired mixtures are then exposed to a set of molecular inputs, which can be interpreted as being keyed to human moves in a game, or as assertions of logical propositions. In nucleic acids-based systems, stemming from DNA computation, these inputs can be seen as generic oligonucleotides. Here, we report using reconfigurable nucleic acid catalyst-based units to build a multipurpose reprogrammable molecular automaton that goes beyond single-purpose 'hard-wired' molecular automata. The automaton covers all possible responses to two consecutive sets of four inputs (such as four first and four second moves for a generic set of trivial two-player two-move games). This is a model system for more general molecular field programmable gate array (FPGA)-like devices that can be programmed by example, which means that the operator need not have any knowledge of molecular computing methods.

Residual DNA in thermostable DNA polymerases – a cause of irritation in diagnostic PCR and microarray assays

In a validation trial of a DNA microarray test for chlamydiae we repeatedly observed false-positive PCR amplicons from truly negative samples and non-template controls. Various PCR tests, microarray hybridization and DNA sequencing, revealed that residual Escherichia coli DNA from thermostable DNA polymerases was the cause of this cross-reaction. A subsequent survey showed that only five out of 23 commercial polymerases were free of E. coli DNA. When designing generic oligonucleotide sequences for PCR and PCR microarray-based assays one should be aware of such possible internal contamination, particularly when the target organism is E. coli.

Unrestricted Hybridization of Oligonucleotides to Structure-Free DNA

The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.

Multigenic Family Analysis among the Encephalitozoon Genus

Encephalitozoon cuniculi is a parasite presenting a wide range of hosts among mammals (including human). Bioinformatic analysis of the genome sequence (11 chromosomes for a haploid genome size of 2.9 Mbp) revealed the presence of multigenic families in a subterminal position on the chromosomes (in 5′ of the rDNA copies on 22 extremities). One of these families was called InterB and clustered together about 30 genes within 10 homology groups. According to the predictions, the typical InterB protein presents in its N‐terminal part a signal peptide, followed by two transmembrane domains. The core of this protein is characterized by an α hypervariable region rich in glutamine and lysine. The C‐terminal region presents several conserved motifs (GGAR, YHL and FQI).A set of specific and generic oligonucleotides was designed to study the InterB genomic repertoire. The migration profile of the generic primers PCR products presents five to six bands. This profile is well conserved for the E. cuniculi isolates as well as for the different Encephalitozoon species. Some polymorphism is evident with amplicons of 300 bp in average. Sequence analysis revealed a higher level of polymorphism, especially in the α region. The InterB repertoire of transcripts was studied by reverse transcription. All InterB genes should be transcribed. There is no known homologue for InterB but, considering the position of the genes, we speculate on the possible role of InterB proteins. Similar multigenic family organizations have been described for proteins involved in pathogenicity of parasites such as Trypanosoma, Plasmodium, Giardia or Pneumocysti.

West Nile virus surveillance: A simple method for verifying the integrity of RNA in mosquito (Diptera: Culicidae) pools.

In a West Nile virus (WNV) -free ecosystem, it is essential to verify the integrity of RNA before concluding that RNA extracted from mosquito specimens is negative for WNV gene sequences. The primary objective of our study was to develop a rapid molecular assay to rapidly screen mosquitoes for the presence of 18S RNA and WNV gene sequences. Mosquitoes, collected from multiple sites on the island of O'ahu, were pooled into groups of 1-50 mosquitoes according to capture site, date, and species. Using primer design software and the GenBank database, generic oligonucleotide primer pairs were designed to amplify mosquito18S rRNA gene sequences from different species. RNA was extracted from mosquito pools, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the presence of mosquito18S rRNA and WNV gene sequences. Three of the seven primer pairs successfully detected 18S rRNA sequences for both Aedes and Culex by RT-PCR, and one primer pair successfully amplified 18S rRNA sequences for 15 different mosquito species. All 64 mosquito pools from 10 different sites on the island of Oahu, Hawaii, were negative for WNV nonstructural protein-5 gene sequences. This simple, one-step RT-PCR method for screening mosquito pools for arboviruses will become an increasingly valuable tool as WNV becomes endemic throughout the Americas.

A universal nucleic acid sequence biosensor with nanomolar detection limits.

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.

Specificity of mammalian Y-box binding protein p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip.

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4,096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4,096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.

Comparison of Complex DNA Mixtures with Generic Oligonucleotide Microchips

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to VD-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.

Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen

AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with &lt; 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy &gt;99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 μL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.

Analysis of Mutations in Oral Poliovirus Vaccine by Hybridization with Generic Oligonucleotide Microchips

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 bp to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.