Detection Of Specific DNA Sequences Research Articles

Intrastrand Photo-Crosslinking of 5-Fluoro-2'-O-methyl-4-thiouridine-Modified Oligonucleotides and Its Implication for Fluorescence-Based Detection of DNA Sequences.

DNA photo-crosslinking reactions occur widely in biological systems and are often used as valuable tools in molecular biology. In this article, we demonstrate the application of an oligonucleotide 5-fluoro-2'-O-methyl-4-thiouridine (FSU)-containing probe for the fluorescent detection of specific DNA sequences. The design of the probe was predicated on studies of agents that could adversely affect its efficiency. The most important of these is the intrastrand photo-crosslinking of single-stranded oligodeoxynucleotides bearing FSU. Our research findings indicate that FSU after photoexcitation can react with nonadjacent bases; specifically, it can react with distant thymine and cytosine residues in the chain, forming fluorescent and nonfluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of the FSU residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of FSU-labeled oligonucleotide probes in the fluorescence-based detection of specific DNA sequences because the creation of a fluorescent intrastrand crosslink could produce a false signal. To overcome this problem, replacing thymidine with deoxyuridine in the FSU-labeled oligonucleotide probe is proposed and tested.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection.

Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

Highly specific and sensitive sandwich-type electrochemiluminescence biosensor for HPV16 DNA detection based on the base-stacking effect and bovine serum albumin carrier platform

The detection of specific DNA sequences and the identification of single nucleotide polymorphisms are important for disease diagnosis. Herein, by combining the high specificity of the base-stacking effect with the high reproducibility of bovine serum albumin (BSA) modified electrodes and the high loading performance of DNA nanoclews (DNA NCs), a novel sandwich-type electrochemiluminescence (ECL) biosensor is reported for the highly specific detection of HPV16 (chosen as the model target). The capture probes are loaded by BSA carrier platforms modified on the gold electrode surface to improve reproducibility. DNA NCs loaded with a large amount of Ru(phen)32+ worked as signal probes. The template probe is composed of the complementary strand of the target and two free nucleic acid anchors at the head and tail. In the presence of the target DNA, the template probes can form stacked base pairs with target, generating high base-stacking energy. This results in the shorter free anchors of template probes being able to bind to the capture and signal probes. This eventually forms a sandwich structure that allows Ru(phen)32+ to be near the electrode surface, producing an ECL signal. There is a linear relationship between the signal and the target concentration range from 10 fM to 100 pM, with a detection limit of 5.03 fM (S/N=3). Moreover, the base-stacking effect has single base recognition ability for base pairs, effectively avoiding false positive signals. The results of this strategy for clinical samples are consistent with classical methods.

Curcumin-capped gold nanorods as optical sensing platform for sequence specific detection of DNA based on their self-assembly

We are reporting curcumin-induced gold nanorods as an optical sensing platform for the detection of sequence-specific DNA target through their self-assembly. The combined effect of eco-friendly reducing agent (i.e., curcumin) and silver nitrate in a basic medium (i.e., pH 10) has been attributed for the formation of small gold nanorods (AuNRs) having approximate length and diameter i.e., 19.7 ± 0.8 nm and 6.0 ± 0.5 nm, respectively, and lower longitudinal surface plasmon resonance (SPR) enable to detect and analyse different biomarkers. Further, for evaluating cellular uptake of as-synthesized AuNRs, the cytotoxicity study has been carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on A549 cells and HEPG2 cell lines, respectively, and shown approximately similar cytotoxicity. Interestingly, as-synthesized optically and electronically active AuNRs based nanobiosensing platform enable to detect sequence-specific DNA targets with low level of detection limit i.e., LOD 8.6 ± 0.15 pM for complimentary target (CT) DNA with higher sensitivity and better selectivity. Finally, this study is suggesting a simplistic bio-mediated approach of tuning the shape and size of AuNRs for sensitive, selective and reliable nanobiosensing platform for sequence-specific DNA detection related to cancer cells.

Facile synthesis of 2D Europium-metal organic frameworks nanosheets for highly efficient electrochemiluminescence in DNA detection

Lanthanide metal organic frameworks (Ln-MOFs) with self-luminous behavior are considered promising emitters of electrochemiluminescence (ECL) due to their unique antenna effects. In this study, the novel 2D Europium-metal organic frameworks (Eu-MOFs) nanosheets were synthesized by simply blending 1, 10-phenanthroline-2, 9-dicarboxylic acid (PDA) and Eu salts at room temperature. When N, N-diethylethylenediamine (DEAEA) was used as the efficient co-reactant, the 2D Eu-MOFs exhibited stronger and steadier ECL performance compared with 2D Europium-metal organic gels (Eu-MOGs) synthesized using similar steps, due to their rigid skeletons and higher crystalline degree, which facilitated electron transfer. In addition, we designed a switching mode ECL biosensor, which was combined with an exonuclease-assisted DNA walker cycling signal amplification strategy, enabled the ultra-sensitive detection of the I27L gene, which was significantly associated with an increased risk of maturity-onset diabetes of the young (MODY). When the target DNA was present, DNA walker recycle amplification occurred driven by exonuclease III (Exo III). Subsequently, the ECL intensity of the system was reduced by introducing the ferrocene-labeled probe (H2-Fc) as a signal quencher. Under the optimal experimental conditions, a wide detection linear range of the target DNA was observed from 1 fM to 10 nM with a low detection limit of 447 aM. This research provided new possibilities for a gentle and simple method to obtain 2D MOFs with stable ECL signals for the sensitive detection of specific DNA sequences.

PCR AND OTHER GENOMIC TECHNIQUES APPLIED TO VETERINARY MEDICINE

The detection of specific DNA sequences by Polymerase Chain Reaction (PCR) has been proven extremely valuable for genetic analysis and diagnosis of a variety of infectious disease pathogens in the veterinary sciences. Genomic techniques applied to veterinary medicine has become fundamental, whether for the detection of pathogens through the use of PCR and its variations or by other techniques that allow the creation and development of recombinant proteins and vaccines for veterinary use. PCR technology has become an indispensable tool in veterinary diagnosis. Other techniques, which play an important prophylactic role for several infectious diseases of veterinary interest, such as recombinant vaccines and sequencing, are important tools in the fight against the most pathogens. In this review, the details of PCR technology are explored, as well as some variations of the technique, such as real-time PCR, as well as other genomic techniques of veterinary interest.

Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection.

Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.

In-situ growth of nitrogen-doped carbonized polymer dots on black phosphorus for electrochemical DNA biosensor of Escherichia coli O157: H7

Sensitive and accurate detection technology for pathogenic bacteria is of great social and economic significance in foodborne disease and food safety. In this paper, a novel portable electrochemical DNA biosensor for the detection of specific DNA sequence of Escherichia coli (E. coli) O157: H7 was constructed. To enhance the performance of the electrochemical sensor, a functionalized nitrogen-doped carbonized polymer dots in-situ grown on few-layer black phosphorus (N-CPDs@FLBP) was synthesized and used as the modifier on the surface of screen-printed electrode. Combining gold nanoparticles as immobilization matrix and methylene blue as electrochemical indicator, the analytical performance of this electrochemical DNA biosensor was evaluated using standard complementary ssDNA sequence in the linear concentration range from 1.0 × 10−19 to 1.0 × 10−6 mol/L with a low detection limit as 3.33 × 10−20 mol/L (3 σ). Furthermore, the portable electrochemical DNA biosensor was proposed based on polymerase chain reaction amplification for the detection of the E. coli O157: H7 genomic DNA from chicken meat, which verified the feasibility for practical samples detection. The research has great theoretical and practical significance for the development of electrochemical biosensor of pathogenic bacteria.

Fluorescent Polymerase Chain Reaction Nanokit for the Detection of DNA Sequence in Single Living Cells.

Here, a fluorescent polymerase chain reaction (PCR) nanokit is established to detect the specific DNA sequence in a single living cell. Different from well-developed protocols to load cell-permeable probes into single cell for recognition, the DNA sequence in a cellular nucleus is sorted into a nanopipette in our strategy. The target DNA sequence is reacted with the PCR kit components in the nanopipette to complete a PCR amplification reaction. SYBR Green prefilled in the nanopipette is intercalated into double-stranded DNA to induce fluorescence emission for real-time detection down to a single copy. An obvious increase in the fluorescence is observed that validates the detection of the target DNA sequence in single living cells. The established real-time fluorescent PCR nanokit could adapt the PCR kit for single cell analysis and thus offers an alternatively general and highly sensitive strategy for the detection of specific DNA sequences in single living cells.

Enhancement effect of 2, 3-dimethyl maleic acid on luminol chemiluminescence reactions and its application in detection of sequence-specific DNA related to hepatitis B virus

2, 3-dimethyl maleic acid (DMMA) was found to enhance luminol-H2O2 chemiluminescent (CL) reactions, among which the strongest enhancement effect was observed by using polyethyleneimine-templated gold nanoclusters (PEI-Au NCs) as the catalyst. With the addition of DMMA, the CL signal of the PEI-Au NCs-catalyzed luminol-H2O2 reaction enhanced about 630-fold, and a flash-type CL profile was obtained. Mechanism studies showed that the luminophore was still 3-aminophthalate anions in the excited state (3-APA*), and superoxide radical (O2·-) played an important role during the CL process. Under the optimized experimental conditions, the lowest concentration of PEI-Au NCs can be detected was 0.168 nM which was 82-fold lower than that without an enhancer. Furthermore, the catalytic activity of biotinylated PEI-Au NCs in the DMMA-enhanced luminol system was similar to PEI-Au NCs, providing a good opportunity for the development of CL bioanalysis platforms using PEI-Au NCs as the label. Thus, the DMMA-enhanced luminol-H2O2 system was applied to the CL detection of sequence-specific DNA related to the hepatitis B virus (HBV) using PEI-Au NCs as the label. The CL platform exhibited linearly enhanced CL response with the increasing amount of target DNA ranging from 0.0025 to 0.5 pmol. As low as 0.002 pmol of HBV DNA could be sensitively detected, which was superior to the previously reported methods.

Self-assembled DNA dendrons as signal amplifiers in a DNA probe-based chemiluminescence assay for enhanced colorimetric detection of short target cDNA.

A simple, reliable, and cost-effective method for nucleic acid detection is of increasing interest in clinical diagnostics of infectious and genetic diseases. Currently, enzyme-mediated methods such as polymerase chain reactions and loop-mediated isothermal amplification are the most widely used methods in the qualitative and quantitative diagnosis of long nucleic acid sequences with high sensitivity. However, a high detection sensitivity for short-length sequences remains a significant challenge because it is difficult for the primers to bind to their sequences. Our previous study demonstrated a simple, enzyme-free, and sequence-specific colorimetric detection of 24-nucleotide short target DNA at room temperature using a developed assay that combines catalytic hairpin assembly (CHA) and enzyme-linked immunosorbent assay (ELISA)-mimicking methods. In this follow-up study, we aim to design and develop DNA-based signal amplifiers, or DNA dendrons, to improve the colorimetric detection of short target cDNA in the CHA-mediated ELISA-mimicking assay. DNA dendrons are highly branched conformations synthesized by the molecular self-assembly of three DNA oligomers. The assay using DNA dendrons demonstrates an enhanced detection sensitivity with the detection of approximately 50 pM of 24-nucleotide short target cDNA, which is a 16.4-fold higher detection limit compared to that obtained without DNA dendrons under the same conditions. Thus, applications of the developed DNA dendrons as an effective signal amplifier in DNA probe-based chemiluminescence assays have the potential to improve the colorimetric detection of short target cDNA with high sensitivity for the diagnosis of different diseases.

Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Applications of Fluorescent in situ hybridization (FISH)

Fluorescent in situ hybridization (FISH) is a molecular technique used for the detection of specific DNA sequences within the chromosome. It relies on the complementary binding between the fluorescently labeled probe and the target sequence. This paper describes how this method was first developed, and the basic principle and the procedure behind it. Furthermore, it covers the basic applications of FISH, including its use in microbiological diagnostics, diagnosis of solid tumors, diagnosis of hematological malignancies, evaluation of sperm and diagnosis of DiGeorge syndrome, along with its applications in plants.

Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

(Invited) Split G-quadruplex-based Strategy for Sensitive Detection of Pathogenic Bacteria

In this talk, I will introduce a simple, sequence-specific DNA detection method, utilizing a fluorescent 2-aminopurine (2-AP) nucleobase analogue-containing split G-quadruplex as the key detection component. In the sensor, the 2-AP-containing G-quadruplex is split into two segments and linked to a target-specific overhang sequence. The separate G-quadruplex sequences form an active G-quadruplex structure only in the presence of a complementary target DNA, which leads to a significant increase in the intensity of fluorescence from the 2-AP fluorophore. This simple, one-step, homogenous assay was successfully employed to detect target DNA with a high selectivity. In addition, the practical applicability of the detection method was demonstrated by its use in analyzing target DNAs in human serum. To the best of our knowledge, this is the first time that an investigation was carried out in which a fluorescent nucleobase analogue was incorporated into a split G-quadruplex structure and this structure was utilized as the foundation for a specific DNA sensor.

Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach

Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA–DNA duplexes formation, by recording ionic current blockades signature of AuNP–α-HL interactions. This approach offers advantages including: (1) a simple to operate platform, producing clear-cut readout signals based on distinct size differences of PNA-induced AuNPs aggregates, in relation to the presence in solution of complementary ssDNAs to the PNA fragments (2) sensitive and selective detection of target ssDNAs (3) specific ssDNA detection in the presence of interference DNA, without sample labeling or signal amplification. The powerful synergy of protein nanopore-based nanoparticle detection and specific PNA–DNA hybridization introduces a new strategy for nucleic acids biosensing with short detection time and label-free operation.

Naked eye Y amelogenin gene fragment detection using DNAzymes on a paper-based device

Nowadays, great efforts are made in developing new technology for rapid detection of specific DNA sequences, for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. Microfluidic paper-based analytical devices are suitable platforms for the development of point of care devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ detection of single strand DNA by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments.

A Methodology for Ultrasensitive Detection of Sequence-Specific DNA or Uracil-DNA Glycosylase Activity.

Ultrasensitive detection of sequence-specific DNA and uracil-DNA glycosylase (UDG) activity shows great practical significance in clinical diagnostic and biomedical studies. Here, a methodology based on a CRISPR/Cas12a system coupled with enhanced strand displacement amplification (E-SDA) was innovatively established for sequence-specific DNA or UDG activity detection. Sequence-specific DNA or DNA primers processed by UDG and Endonuclease IV can initiate E-SDA, generating auxiliary DNA chains, which act as activators to unlock the indiscriminate collateral cleavage activities (trans-cleavage) of the CRISPR/Cas12a. Then, the activated CRISPR/Cas12a, which intrinsically possesses the ability of significant signal amplification, can indiscriminately cleave the added cleavage reporters in the system. Thus, the multistep amplification of the method was obtained. Under the selected experimental conditions, the established method can achieve an actual sensitivity of sequence-specific DNA up to 100 aM within 2.5 h or ultralow UDG activity (3.1×10-5 U/mL) detection within 3.5 h. We believe that the proposed method will have great potential for practical application in ultrasensitive detection of sequence-specific DNA or UDG activity.

Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP).

Human spaceflight endeavors present an opportunity to expand our presence beyond Earth. To this end, it is crucial to understand and diagnose effects of long‐term space travel on the human body. Developing tools for targeted, on‐site detection of specific DNA sequences will allow us to establish research and diagnostics platforms that will benefit space programs. We describe a simple DNA diagnostic method that utilizes colorimetric loop‐mediated isothermal amplification (LAMP) to enable detection of a repetitive telomeric DNA sequence in as little as 30 minutes. A proof of concept assay for this method was carried out using existing hardware on the International Space Station and the results were read instantly by an astronaut through a simple color change of the reaction mixture. LAMP offers a novel platform for on‐orbit DNA‐based diagnostics that can be deployed on the International Space Station and to the broader benefit of space programs.