Detection Of Target DNA Sequences Research Articles

Validation and comparison of four DNA extraction methods for genetically modified organisms analysis: From DNA quality control to quantification of genetically modified content in real‐life samples matrices

AbstractBackgroundThe analysis of genetically modified organisms (GMOs) in food and feed is broadly based on the detection of target DNA sequences using PCR‐based methods. The first step of a typical workflow in a GMO testing laboratory is DNA extraction and purification. Several DNA extraction methods have been described for this purpose so far. Whatever the method adopted, the yield and the quality of the extracted DNA are essential factors for the success of GMO analysis.ResultsThe DNA extraction yield was variable between the different methods, while quality and efficiency appeared comparable. Each method allowed a different yield and a slight correlation between inhibition and the food/feed matrix extracted was observed. Particularly for challenging matrices, the method used for DNA extraction has a remarkable influence on both the quality and quantity of the recovered DNA. Small variations in value % GM content were observed in complex, heterogeneous matrices but not in CRMs and homogeneous matrices.ConclusionsIn this study, the purpose is not to establish the best method of extraction but to examine several parameters that play an important role in GMO detection and verify a possible effect on the quantified GMO percentage. In particular, it was found that the yield and quality of extracted DNA depends on the type of sample, notably the degree of processing, composition, particle size, and the protocol used, especially in relation to sample intake. The slightly different GM content observed in heterogeneous matrices could raise doubt in the case of samples close to the legal threshold.

Electrochemical biosensor for the detection of a sequence of the TP53 gene using a methylene blue labelled DNA probe

The development of an electrochemical biosensor utilising a hairpin DNA probe labelled with methylene blue for the detection of the TP53 gene is described. Structural rearrangement of the hairpin probe into a linear double DNA strand induced different rates of electron transfer that enabled an increase in current upon hybridization. The increase in current observed upon hybridization was studied at different surface probe densities, hybridization times, concentration and length of target DNA sequences. The biosensor selectively detected a single nucleotide polymorphism in the TP53 gene affecting residue 175 of the p53 protein, with the response more pronounced at shorter hybridization times. Detection of target DNA sequences at nanomolar concentrations was achieved and the sensor possessed good operational and storage stability.

Plant Viruses Detection and Diagnosis Based on Polymerase Chain Reaction Techniques: Review

Plant virus diseases result in the loss of billions of dollars annually by limiting plant production quantity and quality in the world. Among different strategies adapted for plant virus disease management, proper diagnosis and detection are the most important and essential strategies for the development of appropriate control measures. The current advanced techniques developed for the detection of plant viruses provided the chance to take practical managemental actions timely. Nowadays one of the most advanced diagnosis methods, polymerase chain reaction (PCR) is used extensively for the detection and identification of plant viruses. PCR is the advanced method that allows the specific amplification and hence detection of target DNA sequences from a mixture of nucleic acid extract in which specific amplification of targeted fragments of a single or a few copies of source DNA material is achieved within a few hours. The PCR method copies each piece of DNA fragments through all the cycles that leading to an exponential doubling of copies over time. Several modifications of PCR methods have been developed to boost the effectiveness of the method in diagnostic settings based on their applications. Reverse transcriptase PCR, immunocapture PCR, Multiplex PCR, co-operational PCR, and real-time PCR are the common and widely used types of PCR variants. Keywords : Detection techniques; PCR; Plant virus DOI: 10.7176/ALST/86-02 Publication date: March 31 st 2021

Multifluorophore DNA Origami Beacon as a Biosensing Platform.

Biosensors play increasingly important roles in many fields, from clinical diagnosis to environmental monitoring, and there is a growing need for cheap and simple analytical devices. DNA nanotechnology provides methods for the creation of sophisticated biosensors, however many of the developed DNA-based sensors are limited by cumbersome and time-consuming readouts involving advanced experimental techniques. Here we describe design, construction, and characterization of an optical DNA origami nanobiosensor device exploiting arrays of precisely positioned organic fluorophores. Two arrays of donor and acceptor fluorophores make up a multifluorophore Förster resonance energy-transfer pair that results in a high-output signal for microscopic detection of single devices. Arrangement of fluorophores into arrays increases the signal-to-noise ratio, allowing detection of signal output from singular biosensors using a conventional fluorescence microscopy setup. Single device analysis enables detection of target DNA sequences in concentrations down to 100 pM in <45 min. We expect that the presented nanobiosensor can function as a general platform for incorporating sensor modules for a variety of targets and that the strong signal amplification properties may allow detection in portable microscope systems to be used for biosensor applications in the field.

Epidemiological Review of Francisella Tularensis: A Case Study in the Complications of Dual Diagnoses.

Introduction:Tularemia is a rare but potentially fatal disease that develops in numerous wild and domestic animals, including lagomorphs, rodents, cats, and humans. Francisella tularensis bacterium, the causative agent of tularemia, was identified by veterinary personnel at Fort Riley, Kansas during a routine post-mortum evaluation of a domestic feline. However, before formal diagnosis was confirmed, the sample was sent and prepared for rabies testing at the Department of Defense (DoD) U.S. Army Public Health Command Central (PHC-C), Food Analysis and Diagnostic Laboratory (FADL). This case report provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. The epidemiologic characteristics of tularemia, F. tularensis as an organism of military interest, potential laboratory management of F. tularensis, and clinical findings on a case of feline tularemia are discussed. It further raises questions as to whether or not dead animals should be treated as sentinels and be pre-screened for select agents, especially in instances of dual diagnoses.Methods:A necropsy was performed on the cat by the Fort Riley veterinarian, DNA extraction and PCR analyses were conducted by FADL microbiologists, histology and immunohistology analyses were conducted by the Kansas State Veterinary Diagnostic Laboratory, and feline tissue and blood were sent to the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) for confirmatory testing and strain identification of tularemia.Results:Tularemia was identified in the spleen of the cat by the Fort Riley veterinarian and during the histological sampling of the spleen by the Kansas State Veterinary Diagnostic Laboratory. A specific subsequent real-time polymerase chain reaction (RT-PCR) in vitro diagnostic detection of target DNA sequences of F. tularensis was conducted by the FADL microbiologists using a Joint Biological Agent Identification and Diagnostic System (JBAIDS) Tularemia Detection Kit to detect a presumptive qualitative result to detect tularemia in feline and blood samples. USAMRIID also performed RT-PCR and identified genomic DNA from F. tularensis Type A, (SPL15.013.02), thus confirming the FADL’s initial presumptive result of F. tularensis. USAMRIID attempted to culture F. tularensis from three samples (swab, feline tissue, and transfer pipette tip), but no growth consistent with F. tularensis was observed on the cysteine heart agar with sheep blood and antibiotics (CHAB) and chocolate (CHOC) plates. Discussions:Our case study of a dual diagnosis of presumptive F. tularensis and possible rabies exposure transmission from a pet cat to its owner provides insight on how veterinarian staff and laboratory personnel can clinically manage esoteric, unexplained, or post-mortum examinations. Our case study also demonstrates the obligation for cooperation between animal health, human health, and public health professionals in the management of zoonotic diseases.

Electrochemical DNA biosensor based on gold nanoparticles and partially reduced graphene oxide modified electrode for the detection of Listeria monocytogenes hly gene sequence

In this study an electrochemical DNA biosensor with gold nanoparticles (AuNPs) and partially reduced graphene oxide (p-RGO) modified electrode was constructed to detect Listeria monocytogenes hly gene sequence. AuNPs were electrodeposited on the surface of carbon ionic liquid electrode (CILE) and then p-RGO was further deposited at controlled electroreduction conditions. On the p-RGO surface unreduced oxygenal groups were present and could covalently interact with the amine group at the 5′-end of probe ssDNA through carbodiimide linkage, and the resulted ssDNA/p-RGO/AuNPs/CILE could be used to hybridize with the target ssDNA sequence. Differential pulse voltammetry was applied to monitor the DNA hybridization with methylene blue as electrochemical indicator. Under the optimal conditions, the electrochemical biosensor could be applied to the detection of target DNA sequence in the concentration range from 1.0×10−13–1.0×10−6mol/L with the detection limit of 3.17×10−14mol/L (3S0/S). The sensor exhibited good stability and selectivity to one or three-base mismatched ssDNA sequences. PCR sample of Listeria monocytogenes hly gene sequence was successfully detected, indicating the real application of this biosensor.

Application of mercury–mediated thymine–base pairs for successful voltammetric detection of HPV 18

Formation of mercury(II)–mediated thymine base pairs (T − Hg(II) − T) can be successfully used for efficiently bonding partially complementary, thymine rich DNA sequences. For the first time this highly selective T − Hg(II) − T connection was used for effective voltammetric detection of specific DNA sequences characteristic for Human Papillomavirus type 18 (HPV 18) without additional labeling step. The detection of target DNA sequence was based on the electrochemical reduction signal of Hg(II) ions bonded with opposite thymine bases (mispairs) in DNA helix. The obtained detection and quantification limits were equaled (1.2±0.2)·10−14 and (7.5±0.1)·10−14M respectively. Furthermore the laser ablation coupled with inductively coupled plasma mass spectrometry (LA–ICP–MS) and circular dichroism (CD) were used as an auxiliary technique to get additional information and confirm the formation of DNA duplex via specific interaction between Hg(II) ions and thymine bases.

Changes in the volume phase transition temperature of hydrogels for detection of the DNA hybridization process.

A simple biosensing platform which involves the application of thermoresponsive hydrogels (p(NIPA-co-AA)) for detection of target DNA sequences is presented. For this aim the hydrogel based on N-isopropylacrylamide grafted with carboxyl groups was modified with H2N-ssDNA via the amide bond. The detection of target DNA sequences was achieved successfully by monitoring the volume phase transition temperature (VPTT). It was found that the dependence between the VPTT and the concentration of the target complementary DNA is linear in the concentration range from 10-12 to 10-6 M. The proposed DNA detection method is characterized by high sensitivity and good reproducibility. The detection limit obtained (∼1 pM) is a substantial improvement over DNA biosensor labelling with tags, because the detection is based on a physical parameter (VPTT). Circular dichroism (CD) and inductively coupled plasma mass spectrometry with laser ablation (LA-ICP-MS) proved that the hybridization process took place in the hydrogel matrix without any restrictions.

Electrochemical Biosensor for Detection of Target DNA Sequence and Single-Base Mismatch Related to Helicobacter Pylori Using Chlorogenic Acid as Hybridization Indicator

An electrochemical DNA hybridization biosensor was developed using a novel electroactive indicator. The DNA hybridization was detected based on the interaction of chlorogenic acid, CGA, with an 18-mer alkanethiol DNA probe (from Helicobacter pylori) self-assembled gold electrode (S1/MCH/AuE) and its hybridization form (S2-S1/MCH/AuE). The conditions were optimized for the immobilization of probe DNA, the exposure of MCH, the accumulation of indicator and the hybridization process. The differential pulse voltammetric response of the accumulated CGA, as an electroactive indicator, on different electrode surfaces was used to assess the DNA hybridization. The results indicate that there is a significant difference between the voltammetric responses of CGA accumulated on the probes hybridized with non-complementary, mismatch and complementary targets. The potential of the developed biosensor is, thus, confirmed in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. In optimum conditions, CGA electrochemical signals had a linear dependence on the concentration of the complementary DNA within the range of 20.0 to 410.0 nM. Finally, a detection limit of 7.2 nM was obtained for the target DNA.

A Genosensor for Point Mutation Detection of P53 Gene PCR Product Using Magnetic Particles

AbstractThe development of an electrochemical genosensor involving DNA biotinylated capture probe immobilized on streptavidin coated paramagnetic beads and microfluidic based platform for the detection of P53 gene PCR product is reported. The novelty of this work is the combination of a sensitive electrochemical platform and a proper microfluidic system with a simple and effective enzyme signal amplification technology, ELISA, for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene sequence. The biosensor has been applied to detect the PCR amplified samples and the results shows that it can discriminate successfully perfect matched DNA from mutant form.

Synthesis of gold microstructures with surface nanoroughness using a deep eutectic solvent for catalytic and diagnostic applications.

We synthesized highly monodisperse gold microparticles (AuMPs) using a deep eutectic solvent (DES) which composed of choline chloride and malonic acid as both a reaction medium and structure-directing agent. These microparticles exhibit distinctive surface nanoroughness and highly defined diameters that can be precisely controlled over a range of a few micrometers under different reductive conditions. The internal and external structures of the particles are thoroughly investigated by electron microscopy, which is further analyzed in association with their optical properties. We also investigate the gold microparticle concentration-dependent catalytic property employing a reductive reaction of 4-nitrophenol to 4-aminopenol as a model system. Importantly, the gold microparticles are densely functionalized with DNA and reversibly assemble with DNA-gold nanoparticle conjugate probes for the colorimetric detection of target DNA sequences, demonstrating that these novel structures can be utilized as platforms that quickly regulate the optical properties of plasmonic nanoparticles for diagnostic applications.

Determination of DNA Hybridization on Gold Nanoparticle Conjugated Polystyrene Particle Thin Film Using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Attenuated total reflectance Fourier transform infrared spectroscopy was used to detect DNA hybridization on a polystyrene conjugated gold nanoparticle thin film. The gold nanoparticles were synthesized on the surface of poly(ethylenimine) coated polystyrene particles by citrate reduction. Single-stranded DNA was then immobilized on the nanoparticle surface via thiol bonding. Ultraviolet-visible spectrometry was used to monitor the conjugation of the nanoparticles on polystyrene particles and the immobilization of a single-stranded DNA probe. The morphology of the polystyrene-gold nanoparticle thin film was characterized using scanning electron microscopy and showed successful conjugation and immobilization. The infrared spectra obtained from the hybridization showed features of DNA structure and peak shifts at 1657 cm−1 compared to the non-complementary DNA due to changes in hydrogen bonding between N-H and C˭O of complimentary bases pairs. The peaks at 1067, 975, 920, and 859 cm−1, which were shifted to lower wavenumbers in the polystyrene-gold nanoparticle probe and target DNA, indicated hydrogen bonding formation between N-H and N of complimentary base pairs. ATR-FTIR spectroscopy provided simple, fast, and portable label-free detection of target DNA sequence on the polystyrene-gold nanoparticle thin film.

Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations

Single molecule assays were performed on streptavidin-β-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule β-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min(-1), which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be -1.32 × 10(-4) ± 0.19 × 10(-4) cm(2)V(-1)s(-1). The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-β-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.

The Role of Fish in Hematologic Cancer

SUMMARY Cytogenetic analysis is now considered a mandatory investigation in the diagnostic work-up of hematologic malignancies. Recurring structural aberrations serve as powerful markers not only for diagnosis and prognosis of these conditions, but also guide the selection of targeted drugs for personalized oncology. The FISH approach is established as an indispensable tool to complement conventional cytogenetics, in addition to basic and clinical research applications. FISH is used to identify specific chromosomal aberrations through the detection of target DNA sequences by fluorescently labeled DNA probes. Multicolor FISH analysis allows the accurate identification of recurring translocations in neoplastic cells by means of genomic probes that flank the breakpoints. This review summarizes the panel of FISH probes for selection and the current utilization of these FISH techniques in unraveling chromosomal aberrations. The niche of FISH analysis is also highlighted. Variant signal patterns of the clinically useful FISH probes for hematologic oncology illustrated here provide useful interpretative reference for molecular pathology laboratories. In addition, the recent application of FISH tests in contributing information on drug targets at the genomic level to support personalized oncology will also be discussed.

Ultra-selective and sensitive DNA detection by a universal apurinic/apyrimidinic probe-based endonuclease IV signal amplification system

A novel signal amplification system that is applicable to any DNA sequence of interest has been developed by using a combination of apurinic/apyrimidinic probe and endonuclease IV. The system allows rapid, highly selective and sensitive detection of target DNA sequences at very low concentrations (10 fmol) or low abundance levels (1%).

Label-free detection of target DNA sequence and single-base mismatch in hepatitis C virus corresponding to oligonucleotide by resonance light scattering technique

A highly sensitive assay to detect sequence-specific DNA by the resonance light scattering (RLS) technique has been developed based on the enhanced RLS intensities of the RLS spectra at 399.5 nm. It was found that Hoechst 33258, when bound to dsDNA and ssDNA in electrolyte solution, displayed different RLS signals. This encouraged us to perform a direct and unlabelled sequence-specific DNA assay. At optimal conditions, this RLS assay can detect complementary target DNA over a range of 4.0 × 10−9–3.8 × 10−7 mol L−1, and the limit of detection was around 1.7 × 10−9 mol L−1. Additionally, the results of experiments showed that different RLS signals reflected a different degree of mismatch between probe DNA and target DNA, and mismatched variants of target DNA with single-base difference can even be well discriminated. Herein, we moved the RLS technique one-step further toward sensitivity, rapidity, simplicity and non-toxicity. The RLS assay results were identified with a fluorescence method, fluorescence polarization assay and atomic force microscope measurement.

Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide

Development of an electrochemical biosensor based on peptide nucleic acid (PNA) probe for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene corresponding oligonucleotide using methylene blue (MB) as an electrochemical indicator is described. The interaction between MB and short sequence of p53, one of the most important tumor suppressor genes due to its dysfunction in the majority of human cancers, was studied by differential pulse voltammety (DPV). Probe modified electrode was prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of gold electrode (GE). The hybridization of PNA probe with target DNA was performed in solution to form PNA–DNA hybrid on the surface of the GE. A significant increase in the reduction signal of MB was observed upon hybridization of the probe with the complementary DNA. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Furthermore, our results confirmed the ability of the sensor to detect single base mismatch in the sample oligonucleotide. The influence of probe concentration on the effective discrimination against noncomplementary sequence and point mutation was also investigated. Diagnostic performance of the biosensor is described and the detection limit is found 6.82 × 10 −10 M. The electrochemical impedance spectroscopy was also employed to further investigate the sensor function.

Electrochemical detection and discrimination of single copy gene target DNA in non-amplified genomic DNA

A sensitive, fast and inexpensive method for direct electrochemical detection of target DNA sequences in non-amplified genomic DNA samples is described. Hybridization detection relies on the alteration in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. Initially, the method was tested to detect target DNA on low cycle number PCR amplicons. Having obtained promising detection results from only 5 cycles product, the feasibility of target sequence detection in extracted genomic DNA without PCR amplification, but with the vortex mediated fragmentation of the large genomic DNA into small pieces was examined. Experimental variables affecting the efficiency of sensor were investigated. Detection experiments with various non-complementary genomic DNAs as well as a proper probe, non-specific with respect to all genomic samples confirmed the excellent selectivity of the approach. The sensitivity of the method for analyzing the vortex mediated fragmentized genomic DNA samples is estimated to be approximately of 0.58ng/μl.

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.

Affinity Sensing for Transgenes Detection in Antidoping Control

Sports authorities fear that a new form of doping called gene doping, based on the misuse of gene therapy, represents an emerging important problem and so far no methods are available for detecting it. The World Anti-Doping Agency (WADA) has included since 2003 for the first time gene doping methods in the "Prohibited List of Substances and Methods", thus detection of this new form of doping is challenging for analytical chemists. In this work, we apply affinity-based biosensors (ABBs), in particular DNA piezoelectric sensing, for detection of target DNA sequences selected as transgenosis markers. In this work, two sequences widely used in transgenosis experiments have been identified as markers: the enhanced green fluorescence protein (EGFP) gene and the promoter of Cytomegalovirus (CMV). The biosensors are characterized in their analytical performances using synthetic oligonucleotides and amplified DNA obtained from purified plasmid used as a template. Finally they have been applied to transgenic human cell cultures (human embryonic kidney HEK-EGFP), transformed with the same plasmid and carrying the target markers. This represents the closest human real matrix available for our transgenes.