A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Spiky gold shells on magnetic particles for DNA biosensors.

Combined separation and detection of biomolecules has the potential to speed up and improve the sensitivity of disease detection, environmental testing, and biomolecular analysis. In this work, we synthesized magnetic particles coated with spiky nanostructured gold shells and used them to magnetically separate out and detect oligonucleotides using SERS. The distance dependence of the SERS signal was then harnessed to detect DNA hybridization using a Raman label bound to a hairpin probe. The distance of the Raman label from the surface increased upon complementary DNA hybridization, leading to a decrease in signal intensity. This work demonstrates the use of the particles for combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step.

Core-shell assay based aptasensor for sensitive and selective thrombin detection using dark-field microscopy.

In this work, we developed a robust and ultrasensitive bio-sensor based on the target-aptamer recognition strategy and microscopic enumeration of gold nanoparticles (AuNPs) using dark field microscopy (DFM). The aptasensor with a core-shell structure consisting of a magnetic bead (MB), aptamer and AuNPs was fabricated by complementary hybridization of the DNA probe on the AuNPs surface to the aptamer coupled to the MB. Upon addition of the target molecule, the strong interaction between the aptamer and the target molecule, thrombin, results in the release of the AuNPs from the MB. The quantities of thrombin is therefore linearly correlated to the number of the released AuNPs, which can be digitally counted using DFM. To demonstrate the feasible use of the aptasensor for target detection, thrombin was evaluated as the model target. The limit of detection was determined to be 2.54 fM with dynamic range of 6 fM-100 fM. When the concentration of thrombin exceeded 100 fM, the counted number of AuNPs didn't correlate linearly to molecules of thrombin anymore, as the nanoparticles aggregated partly due to high concentration. However, the color of the solution changes to purple and the concentration of free AuNPs can be conveniently quantified by UV-Vis spectroscopy for up to 100 nM. It is noteworthy that our aptasensor is very easy to operate and requires neither complex isolation and amplification processes nor expensive instruments and consumables. Furthermore, this strategy can be easily generalized to other targets by replacing the corresponding aptamers and show great potential for the detection of biomarkers in clinical samples.

A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection.

Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/µL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/µL and a regression coefficient (R(2)) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

Analysis of receptor tyrosine kinase gene amplification on the example of FGFR1.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

[Molecular technologies in detecting sensitive and isoniazid-resistant Mycobacterium tuberculosis].

Two types of techniques for detection of single nucleotide polymorphism in 315 codon of katG gene of MTB are developed. Isoniazid resistance of MTB is associated with point mutations in the mentioned codon. Two primer sets with additional competitive blocking primer containing 3'-terminal phosphate group (for elimination of unspecific amplification) allow detecting the most frequent point mutations AGC --> ACC and AGC --> AGA in 315 codon of katG gene. PCR with primer set of two primers one of which contains five LNA-monomers allows to determine an occurrence of any type from six known mutations in 315 codon of katG gene, i.e. to differentiate wild type and isoniazid-resistant MTB. Purity and structure of 17 bp long primers with LNA-modified nucleotides were characterized by time-of-flight MALDI-mass spectrometry. Duplex of 17 bp length formed by two complementary oligonucleotides with LNA-monomers was studied using melting.

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups.

We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

Fluorescent DNA probes: study of mechanisms of changes in spectral properties and features of practical application.

Spectral data and nucleic acid complex formation properties for more than 30 both newly synthesized and widely used fluorescent nucleotide-specific compounds of various classes have been analyzed. These include phenylbenzene, bisbenzimidazole, psoralen, angelicin, tetrahydrocarbazole, oxophenoxazine, and others. The main rules of a generalized model adequately explaining changes in fluorescent properties of synthetic, low molecular weight nucleotide-specific dyes depending on their chemical structure, mode of interaction with substrate, properties of assay medium, etc. are proposed. Fluorescent nucleotide-specific dyes have been originally used in newly developed methods for: express evaluation of "generalized microbial dissemination" of liquid media; evaluation of possible genotoxic effects of various foodstuffs, pharmaceutical drugs, hazardous environmental factors (including their combined effects on living organisms), etc.

Microarray analysis of cardiovascular diseases.

Microarray analysis is a powerful technique for high-throughput, global transcriptonomic profiling of gene expression. It holds great promise for analyzing the genetic and molecular bases of cardiovascular diseases and various other complex diseases and permits the analysis of thousands of genes simultaneously, both in diseased and nondiseased tissues and/or cell lines. Microarrays or microchips are made by depositing spots of DNA or oligonucleotides representing thousands of genes on a solid support such as a coated glass surface, and can allow the comparison of gene expression patterns in any two samples. Total RNA is isolated from the tissue or cells of interest, converted to cDNA and then cRNA labeled with biotin, and hybridized to the chips. Hybridization signals are then quantified and compared among different samples. We used oligonucleotide microarrays to obtain an unbiased assessment of expression levels of thousands of genes simultaneously in normal and diseased coronary arteries. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues, and 49 of them represent new linked genes for coronary artery disease. These studies can generate novel hypotheses relating to the pathologies of disease and further studies with animal models, molecular biology, cell biology, and biochemistry will validate these hypotheses and provide novel insights into the pathogenesis of disease.

Graphite-epoxy composites as a new transducing material for electrochemical genosensing.

The use of a rigid carbon-polymer composite material as an electrochemical transducer in hybridisation genosensors is reported. Graphite-epoxy composites (GEC) have an uneven surface where DNA can be adsorbed using a simple dry-adsorption procedure. Single-stranded-DNA binds strongly to GEC in a way that prevents the strands from self-associating, while permitting hybridisation with complementary DNA. Hybridisation has been detected through biotin-streptavidin interaction using a streptavidin conjugated to horseradish peroxidase. Non-specific adsorption onto GEC is almost non-existent even when the surface has not been treated by blocking reagents. The analytical signal obtained was higher when compared with other electrochemical genosensors. Results can be achieved in 150 min, and the detection limit is in the order of fmol. Additionally, surface regeneration is possible using a simple polishing procedure, allowing for multiple use. The new genosensor based on GEC fulfils the requirements desired for these devices: ease of preparation as dry-adsorption of DNA is very simple and easily automated, robustness, sensitivity, low cost of production, ease of miniaturisation and simple use and fast response. Additionally, it can be used for field measurements and can be produced as a genosensor kit. Also, this material can be implemented for screen-printing procedures for the mass production of genosensors. The utility of the genosensor based on GEC is also illustrated with the detection of a sequence related to novel determinant of beta-lactamase resistance in Staphylococcus aureus.

Controlled expression of an rpoS antisense RNA can inhibit RpoS function in Escherichia coli.

We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents.

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.

Reproducible and inexpensive probe preparation for oligonucleotide arrays.

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

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

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

Quantitative and sensitive northern blot hybridization using PCR-generated DNA probes labeled with digoxigenin by nick translation.

Northern blot hybridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern blot hybridization was performed using the DIG-labeled DNA probes, and the signals were detected by means of a chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern blots with low background. Densitometric analysis of the blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.

Growth hormone receptors in hypothalamic and extra-hypothalamic tissues.

Central GH receptors (GHR) have been identified in hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains. Plasma membranes of the rabbit brain demonstrated specific saturable high-affinity, low-capacity binding sites for 125I-labelled GH. RNA extracted from hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains contained mRNA that hybridized with a cDNA probe for the rabbit liver GHR. This transcript was of a similar size to the major GHR mRNA moiety in rabbit liver. The expression of these moieties was age related, and higher in adult than in neonatal animals.

[Evaluation of the relative effectiveness of hybridization probes prepared by action of hydrazides of biotin derivatives on DNA]. / Otsenka otnositel'noi éffektivnosti gibridizatsionnykh zondov, poluchennykh deistviem gidrazidov proizvodnykh biotina na DNK.

Efficiencies of biotinylated DNAs as hybridisation probes in a model system of non-radioactive detection were compared. Probes were obtained by interaction of single-stranded DNA and each of four different hydrazides of biotin derivatives. The most sensitivity in detection of complementary target was obtained using (biocytin hydrazide)-treated DNA. Relations between hydrazide structures and sensitivity of biotinylated probes are discussed.

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes.

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.