Hybridization Of DNA Probes Research Articles

Synthesis of Ag2S–Ag Nanoprisms and Their Use as DNA Hybridization Probes

A simple synthetic route to prepare Ag(2) S-Ag nanoprisms consists of the facile addition of Na(2) S to a solution of triangular Ag nanoprisms. The resulting Ag(2) S-Ag nanoparticles are more stable in solution than the original Ag nanoprisms, and two surface plasmon resonance (SPR) bands of the original Ag nanoprisms still remain. In addition, the SPR bands of the Ag(2) S-Ag nanoprisms are tunable over a wide range. The Ag(2) S-Ag nanoprisms can be directly bioconjugated via well-established stable Ag(2) S surface chemistry with readily available sulfur coupling agents. The nanoprisms are used in the hybridization of functionalized oligonucleotides, and show promise as probes for future biosensing applications.

Patterned Immobilization of Biomolecules on a Polymer Surface Functionalized by Radiation Grafting

Patterned graft polymerization of a functional monomer on a hydrophobic polymer surface was proposed for biomolecule patterning. A poly(vinylidene fluoride) (PVDF) film surface was selectively activated by ion implantation through a pattern mask and acrylic acid (AA) was then graft polymerized onto the activated regions of the PVDF surfaces. The peroxide concentration on the implanted surface depended on the fluence, which had a considerable effect on the grafting degree of AA. Afterwards, amine-functionalized biotin and probe DNA were immobilized on the poly(acrylic acid)-grafted regions of the PVDF surfaces. Specific binding of biotin with streptavidin and hybridization of probe DNA with complimentary DNA proved successful protein and DNA patterning and well-defined 50 microm dot-type patterns of the streptavidin and DNA were obtained. These results confirmed the potential of this strategy for patterning of various biomolecules.

Label free DNA detection based on gold nanoparticles quenching fluorescence of Rhodamine B

A novel and sensitive label free DNA detection method using gold nanoparticles (GNPs) and Rhodamine B (RB) has been developed. The assay is based on the following two properties. One is the different adsorption properties of single-stranded and double-stranded DNA on GNPs in colloidal solution. The other is the different quenching ability of aggregated GNPs and dispersed GNPs on RB. Un-aggregated GNPs could effectively quench the fluorescence of RB. However, the quenching ability greatly decreases after GNPs aggregated. The hybridization of probe DNA and target DNA is monitored by the fluorescence detection after the RB is added to the solution. Under the optimal experimental conditions, the detection limit of this assay is 2.9×10−13molL−1.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.

Analysis of microRNA signatures using size-coded ligation-mediated PCR

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.

Human papillomavirus genotypes and their prevalence in a cohort of women in Trinidad.

Human papillomavirus (HPV) genotypes and their relative prevalences were determined in a cohort of 310 sexually active women in Trinidad, West Indies. Cervical samples were collected with Ayre's spatulas and endocervical brushes. Samples were used for the conventional Papanicolaou test and for determining HPV genotypes by amplification of a section of the viral L1 gene, followed by DNA sequencing and probe hybridization. HPV infections were identified in 126 of 310 (40.6%) women. Of them, 83 (65.8%) were infected with high-risk HPV, 16 (12.7%) with low-risk HPV, and 27 (21.4%) with HPV types of unknown risk. HPV 52 (12.7%) was the most frequently occurring high-risk type, followed by HPV 66 (10.3%), HPV 16 (9.5%), and HPV 18 (8.6%). High-risk types HPV 16 and HPV 66 were each found in 3 (20.0%) and HPV 18 was found in 1 (6.6%) of the 15 women with abnormal cytology. Cervical HPV prevalence and heterogeneity of HPV genotypes are high in this Trinidad cohort. The relative importance of HPV genotypes in the development of cervical lesions needs further investigation in Trinidad in order to better understand the epidemiology of HPV infections as well as to determine the role of HPV testing in the screening, prevention, and control of cervical cancer. This pilot study provided important information on the prevalence of HPV genotypes, which will be used in future nationwide studies.

Synthesis of mono-, di-, tri- and tetracarboxy azaphthalocyanines as potential dark quenchers

Mono-, di-, tri- and tetracarboxy-substituted metal-free azaphthalocyanines (AzaPc) were synthesized from 5,6-bis(diethylamino)pyrazine-2,3-dicarbonitrile and 6-(5,6-dicyano-3-(diethylamino)pyrazin-2-ylamino)hexanoic acid using a statistical condensation approach. AzaPc bearing eight diethylamino peripheral substituents was also isolated from the mixture. Analysis of the distribution of congeners in the statistical mixture using optimized HPLC method (Phenomenex Synergy RP Fusion column, acetonitrile/tetrahydrofuran/water (pH 5.5) 50:20:30) was performed. The analysis showed optimal ratios of starting materials to be 3:1 for AAAB, 1:3 for ABBB and 1:1 for AABB/ABAB types of the congeners. The distribution of the congeners corresponded well with calculated values indicating similar reactivity of both starting materials and no sterical constraint between adjacent isoindole units in the AzaPc ring. All investigated AzaPc showed no fluorescence, extremely low singlet oxygen quantum yields ( Φ ∆ < 0.005) in monomeric form and strong absorption in a wide range from 300 nm to almost 700 nm. Such properties are highly promising for future investigation of these compounds as dark quenchers of fluorescence in DNA hybridization probes.

Dynamic chemistry for enzyme-free allele discrimination in genotyping by MALDI-TOF mass spectrometry

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease origins and differences in drug responses. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or alternatively by the hybridization of DNA probes. MALDI-TOF mass spectrometry (MS) has been widely used as a read-out tool. We developed a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping. In a blind trial, peptide nucleic acid (PNA) probes and aldehyde-modified nucleobases were employed to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. MALDI-TOF MS reported the resulting allele-specific products. Enzyme-free dynamic chemistry permitted the genotyping of twelve individuals for the ΔF508 (indel) and G551D (SNP) mutations. No false positives or negatives were observed, and analysis could be performed in both singleplex and duplex formats. Dynamic chemistry provides a distinct method of allele discrimination with certain advantages over those reported previously. Although PCR amplification is required, the analysis stage is performed without enzymes, and there is no need for the stringent optimization and control of conditions associated with hybridization-based methods of allele discrimination.

Development of Reflective Biosensor Using Fabrication of Functionalized Photonic Nanocrystals

Photonic crystals (PCs) are periodic dielectric structures that have a band-gap that forbids propagation of a certain range of wavelengths of light. This property enables control of light with remarkable facility by modification of the band-gaps and produce effects that are impossible with conventional optics. Using chemically functionalized PCs, where the chemical functional group consists of amine and carboxyl group, in conjunction with a biomolecular probe material, the detection of pathogens and viral disease is possible, indicated by the shift in wavelength signal. Moreover, this system using the bioinspired PCs allows specific target detection in biosensor chip fields through control of the PCs. In this study, we demonstrated that two bacterial pathogens (Fusobacterium necrophorum and Acinetobacter baumannii) causing sepsis were detected by DNA-probe hybridization and a severe acute respiratory syndrome coronavirus was detected by antigen-antibody interaction using the functional PCs. Optical readout with the integrated sensor detecting the signals from PCs, allows for low cost and robust readout of resonance peak shift. This biosensor system using the functional PCs on the photonic crystal-fabricated chip can efficiently and effectively detect various targets, and be easily prepared with high productivity and economic property.

Sensitive detection of Salmonella with fluorescent bioconjugated nanoparticles probe

A sensitive and specific method for detecting foodborne pathogen, with Salmonella as model analyte, has been developed basing upon DNA hybridization and fluorescent bioconjugated nanoparticles probe. The indicator probe was designed through bioconjugating the Salmonella sequence-specific oligonucleotide with the fluorescent Ru ( bpy ) 3 2 + -doped silica nanoparticles that were prepared by a microemulsion method. Through a sandwich-type DNA hybridization procedure, the target Salmonella DNA was captured and the indicator probe was assembled onto solid matrix so that the Salmonella DNA can be measured by the fluorescent signals of assembled indicator probes. Under optimal conditions, the calibration graph for detection of target Salmonella DNA is linear over the range of 10–10,000 fmol/L with a detection limit of 3 fmol/L. The proposed method was also applied to detect Salmonella in mixed bacteria sample. It gives the detection limit of 30 cfu/mL. The method offers a potential resolution for simple, rapid and sensitive detection of foodborne pathogens in food, clinical and environmental samples.

Environmental nitration processes enhance the mutagenic potency of aromatic compounds

This work is an attempt to establish if aromatic nitration processes are always associated with an increase of genotoxicity. We determined the mutagenic and genotoxic effects of Benzene (B), Nitrobenzene (NB), Phenol (P), 2-Nitrophenol (2-NP), 2,4-Dinitrophenol (2,4-DNP), Pyrene (Py), 1-Nitropyrene (1-NPy), 1,3-Dinitropyrene (1,3-DNPy), 1,6-Dinitropyrene (1,6-DNPy), and 1,8-Dinitropyrene (1,8-DNPy). The mutagenic activities were evaluated with umuC test in presence and in absence of metabolic activation with S9 mix. Then, we used both cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes on human lymphocytes in order to evaluate the genotoxic effects. Analysis of all results shows that nitro polycyclic aromatic hydrocarbons (PAHs) are definitely environmental genotoxic/mutagenic hazards and confirms that environmental aromatic nitration reactions lead to an increase in genotoxicity and mutagenicity properties. Particularly 1-NPy and 1,8-DNPy can be considered as human potential carcinogens. They seem to be significant markers of the genotoxicity, mutagenicity, and potential carcinogenicity of complex PAHs mixtures present in traffic emission and industrial environment. In prevention of environmental carcinogenic risk 1-NPy and 1,8-DNPy must therefore be systematically analyzed in environmental complex mixtures in association with combined umuC test, CBMN assay, and FISH on cultured human lymphocytes. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Comparative FISH analysis of C-positive blocks of centromeric chromosomal regions of pygmy wood mice Sylvaemus uralensis (Rodentia, Muridae)

The composition and homology of centromeric heterochromatin DNA has been compared in representatives of the Asian race and two chromosomal forms (Eastern European and Southern European) of the European race of the pygmy wood mouse Sylvaemus uralensis by means of in situ hybridization with metaphase chromosomes of microdissection DNA probes obtained from centromeric C-blocks of mice of the Southern European chromosomal form and the Asian race. Joint hybridization of both DNA probes yielded all possible variants of centromeric regions in terms of the presence of repetitive sequences homologous to those of some or another dissection region, which indicates a diversity of centromeric regions differing in DNA composition. However, most variations of the fluorescent in situ hybridization (FISH) patterns are apparently related to quantitative differences of repetitive elements of the genome. Experiments with the DNA probe obtained from the genome of the Southern European form of the pygmy wood mouse have shown that the number of intense FISH signals roughly corresponds to the number of large C-segments in representatives of the European race, which is characterized by a large amount of the centromeric C-heterochromatin in the karyotype. However, intense signals have been also detected in experiments on hybridization of this probe with chromosomes of representatives of the Asian race, which has no large C-blocks in the karyotype; thus, DNA sequences homologous to heterochromatic ones are also present in nonheterochromatic regions adjacent to C-segments. Despite the variations of the numbers of both intense and weak FISH signals, all chromosomal forms/races of S. uralensis significantly differ from one another in these characters. The number of intense FISH signals in DNA from the samples of pygmy wood mice from eastern Turkmenistan (the Kugitang ridge) and southern Omsk oblast (the vicinity of the Talapker railway station) was intermediate between those in the European and Asian races, which is apparently related to a hybrid origin of these populations (the hybridization having occurred long ago in the former case and recently in the latter case).

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

BackgroundFrom shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism.FindingsThe inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration.ConclusionsOur validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

Chlamydia Prevalence Among Women and Men Entering the National Job Training Program: United States, 2003–2007

To analyze 5-year prevalence trends in Chlamydia trachomatis infections among high-risk young men and women aged 16 to 24 years entering the National Job Training Program, where universal screening is required. Entrance exams conducted in over 100 National Job Training Program centers from 2003 to 2007 were considered. Women provided cervical specimens tested using either a DNA hybridization probe (PACE 2, Gen-Probe, San Diego, CA) or a strand displacement amplification test (SDA, BD ProbeTec ET, Becton-Dickinson, Sparks, MD). In the absence of a pelvic exam, urine specimens were tested using SDA. PACE 2 testing was performed predominately from 2002 to 2005; from 2005 to 2007, SDA was used. All male testing was conducted using SDA on urine specimens. Chlamydia prevalence trends were assessed for women and men, using logistic regression models. Adjusted odds ratios (AOR), 95% confidence intervals (CI), and P-values were calculated. Approximately 15,000 women and 30,000 men were screened annually for chlamydia. Among both sexes, adjusted prevalence declined significantly from 2003 to 2007. In 2003, crude prevalence among women was 9.9%; in 2007, prevalence was 13.7%. However, after controlling for covariates, including increasingly sensitive tests, the model indicated a significant declining prevalence trend (AOR: 0.95, CI: 0.93-0.97, 4.6% decrease in odds per year). Among men, crude prevalence in 2003 was 8.4%; in 2007, prevalence was 8.3%; after controlling for possible confounding, a significant decline in prevalence was also detected (AOR: 0.98, CI: 0.96-0.99, 1.9% decrease in odds per year). In a relatively stable, high-risk population of young women and men, adjusted chlamydia prevalence declined from 2003 to 2007. Test technology plays a critical role in interpreting rates and should be considered whenever chlamydia rates are examined.

Advanced microfluidic packaging for molecular diagnostics

STMicroelectronics has teamed up with Boehringer Ingelheim microParts GmbH to develop the In-Check™ Lab-on-Chip microfluidic disposable cartridge. In-Check™ is a ST proprietary platform dedicated to the in-vitro molecular diagnostics, e.g. biological analysis based on nucleic acid targets such as DNA, RNA. Its first generation has been released to commercial applications such as virus or bacteria borne infectious diseases. The second generation described herein will further enhance the platform customer experience by means of an innovative design of its disposable component. Indeed the new format represents a substantial step forward in the system integration and easy-of-use. This advanced microfluidics package enables fully automated application protocols such as analyte and reagents input, management and disposal. In-Check™ cartridge is a plastic-based self-contained embodiment which integrates a variety of design elements and components, including liquid loading and waste reservoirs, connecting micro-channels, two sets of valves one set to tight-seal biochemical reactors and the other set for fluid routing, hydrophobic membranes, fluorescent read-out window. Such microfluidic platform married with integrated Micro Electo Mechanical System (MEMS) device and software algoritms provides a highly flexible system to run complex biological assays as RNA Reverse Transcription (RT), DNA Polimerase Chain Reaction (PCR), Probe Hybridization and Detection. This paper will present the disposable product concept, key components and functionality together with the design and manufacturing challenge.

Red–white–blue emission switching molecular beacons: ratiometric multicolour DNA hybridization probes

Molecular beacons with two fluorophores derived from ethynyl pyrene and ethynyl nile red exhibit red emission in the hairpin that changes to blue through white upon binding to the target sequence with a dramatic shift of approximately 225 nm.

Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.

A non-labeled DNA biosensor based on light addressable potentiometric sensor modified with TiO2 thin film

Titanium dioxide (TiO(2)) thin film was deposited on the surface of the light addressable potentiometric sensor (LAPS) to modify the sensor surface for the non-labeled detection of DNA molecules. To evaluate the effect of ultraviolet (UV) treatment on the silanization level of TiO(2) thin film by 3-aminopropyltriethoxysilane (APTS), fluorescein isothiocyanate (FITC) was used to label the amine group on the end of APTS immobilized onto the TiO(2) thin film. We found that, with UV irradiation, the silanization level of the irradiated area of the TiO(2) film was improved compared with the non-irradiated area under well-controlled conditions. This result indicates that TiO(2) can act as a coating material on the biosensor surface to improve the effect and efficiency of the covalent immobilization of biomolecules on the sensor surface. The artificially synthesized probe DNA molecules were covalently linked onto the surface of TiO(2) film. The hybridization of probe DNA and target DNA was monitored by the recording of I-V curves that shift along the voltage axis during the process of reaction. A significant LAPS signal can be detected at 10 micromol/L of target DNA sample.

Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis

Bacterial endophthalmitis is a serious but uncommon intraocular infection which frequently results in vision loss. Early diagnosis and appropriate therapy are associated with better visual outcome. Conventional microbiological methods are currently used for microbiological characterization of eyes with suspected endophthalmitis. However, the sensitivity of bacterial detection from aqueous and vitreous humor using microbiology techniques is poor, and time-consuming to confirm the results. The application of molecular methods enhances significantly laboratory confirmation of bacterial endophthalmitis, demanding a shorter time to draw a definitive result and thereby promoting the early initiation of a more specific therapy to limit the empirical or unnecessary use of broad-spectrum antibiotics. PCR-based techniques, including post-PCR methods such RFLP, DNA probe hybridization and DNA sequencing have been successfully used for the diagnostic elucidation of clinically suspected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories.