Conventional DNA Research Articles

Three-tiered authentication of herbal traditional Chinese medicine ingredients used in women's health provides progressive qualitative and quantitative insight.

Traditional Chinese Medicine (TCM) herbal products are increasingly used in Europe, but prevalent authentication methods have significant gaps in detection. In this study, three authentication methods were tested in a tiered approach to improve accuracy on a collection of 51 TCM plant ingredients obtained on the European market. We show the relative performance of conventional barcoding, metabarcoding and standardized chromatographic profiling for TCM ingredients used in one of the most diagnosed disease patterns in women, endometriosis. DNA barcoding using marker ITS2 and chromatographic profiling are methods of choice reported by regulatory authorities and relevant national pharmacopeias. HPTLC was shown to be a valuable authentication tool, combined with metabarcoding, which gives an increased resolution on species diversity, despite dealing with highly processed herbal ingredients. Conventional DNA barcoding as a recommended method was shown to be an insufficient tool for authentication of these samples, while DNA metabarcoding yields an insight into biological contaminants. We conclude that a tiered identification strategy can provide progressive qualitative and quantitative insight in an integrative approach for quality control of processed herbal ingredients.

Proximity hybridization induced molecular machine for signal-on electrochemical detection of α-synuclein oligomers

α-synuclein oligomer is a marker of Parkinson's disease. The traditional enzyme-linked immunosorbent assay for α-synuclein oligomer detection is not conducive to large-scale application due to its time-consuming, high cost and poor stability. Recently, DNA-based biosensors have been increasingly used in the detection of disease markers due to their high sensitivity, simplicity and low cost. In this study, based on the DNAzyme-driven DNA bipedal walking method, we developed a signal-on electrochemical sensor for the detection of α-syn oligomers. Bipedal DNA walkers have a larger walking area and faster walking kinetics, providing higher amplification efficiency compared to conventional DNA walkers. The DNA walker is driven via an Mg2+-dependent DNAzyme, and the binding-induced DNA walker will continuously clamp the MB, resulting in the proliferation of Fc confined near the GE surface. The linear range and limit of detection were 1 fg/mL to 10 pg/mL and 0.57 fg/mL, respectively. The proposed signal-on electrochemical sensing strategy is more selective. It will play a significant role in the sensitive and precise electrochemical analysis of other proteins.

Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling.

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays)or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.

Nomenclatural standards and genetic certificates for apple-tree cultivars developed at the North Caucasian Federal Scientific Center of Horticulture, Viticulture, wine-making

Background. The development of genetic certificates and nomenclatural standards for newly released cultivars of Malus domestica Borkh. (apple tree) ensures an opportunity to preserve the original and authentic genetic and morphological information on domestic cultivars promising for the south of Russia. The purpose of this study was to develop genetic certificates and nomenclatural standards for 7 apple-tree cultivars developed at the North Caucasian Federal Scientific Center of Horticulture, Viticulture, Wine-making (NCFSCHVW), five of which were released jointly with the All-Russian Research Institute of Fruit Crop Breeding (VNIISPK).Materials and methods. The materials included apple-tree cultivars resistant or immune to scab (Venturia inaequalis (Cooke) G. Winter) from the NCFSCHVW collection stock. Conventional herbarization and DNA fingerprinting methods were used. For cultivar certification, an improved technique of DNA sampling and 12 microsatellite markers were applied.Results. Unique DNA profile certificates and nomenclatural standards were produced for 7 apple-tree cultivars: ‘Vasilisa’, ‘Karmen’, ‘Krasny Yantar’, ‘Soyuz’ and ‘Rassvet’ (with the Rvi6 gene) released jointly by the NCFSCHVW and VNIISPK, plus ‘Zolotoye Letneye’ and ‘Feya’ developed at the NCFSCHVW. The apple-tree herbarium specimens were placed for perpetual storage in the WIR Herbarium of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR).Conclusion. The results obtained are promising for identification and authenticity verification of new domestic apple-tree cultivars during their reproduction and cultivation.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

Standard addition method for rapid, cultivation-independent quantification of Legionella pneumophila cells by qPCR in biotrickling filters.

Cultivation-independent molecular biological methods are essential to rapidly quantify pathogens like Legionella pneumophila (L. pneumophila) which is important to control aerosol-generating engineered water systems. A standard addition method was established to quantify L. pneumophila in the very complex matrix of process water and air of exhaust air purification systems in animal husbandry. Therefore, cryopreserved standards of viable L. pneumophila were spiked in air and water samples to calibrate the total bioanalytical process which includes cell lysis, DNA extraction, and qPCR. A standard addition algorithm was employed for qPCR to determine the initial concentration of L. pneumophila. In mineral water, the recovery rate of this approach (73%-134% within the concentration range of 100-5000 Legionella per mL) was in good agreement with numbers obtained from conventional genomic unit (GU) calibration with DNA standards. In air samples of biotrickling filters, in contrast, the conventional DNA standard approach resulted in a significant overestimation of up to 729%, whereas our standard addition gave a more realistic recovery of 131%. With this proof-of-principle study, we were able to show that the molecular biology-based standard addition approach is a suitable method to determine realistic concentrations of L. pneumophila in air and process water samples of biotrickling filter systems. Moreover, this quantification strategy is generally a promising method to quantify pathogens in challenging samples containing a complex microbiota and the classical GU approach used for qPCR leads to unreliable results.

PH-Controlled Resettable Modular DNA Strand-Displacement Circuits.

Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

Conventional net tow versus environmental DNA for metabarcoding-based analysis of plankton-environment interactions in polluted aquatic ecosystems

Human societal development has caused rapid degradation of freshwater ecosystems, resulting in significant biodiversity decline globally. The conservation of aquatic ecosystems, particularly the management of biodiversity in urban areas, has become an increasingly significant issue for ensuring the sustainability of these ecosystems. The restoration of degraded aquatic ecosystems relies on a comprehensive understanding of the interactions between biological communities and multiple stressors derived from human activities. Environmental DNA (eDNA)-based methods have emerged as effective tools for dissecting complex community-environment interactions. Nevertheless, potential causes for technical disparities and conclusions drawn from conventional sampling versus eDNA-based methods remain inadequately evaluated. In this study, we conducted a comprehensive survey of plankton communities along the environmental gradient of the North Canal River in Northern China to assess causes and consequences for any disparities between conventional plankton net tow and eDNA sampling for metabarcoding-based analysis of plankton-environment interactions. Our findings demonstrated that eDNA-metabarcoding was effective for characterizing both biodiversity and geographical distribution. Both methods exhibited general consistency in elucidating community structure and identifying the key environmental factors influencing geographical distribution of both zooplankton and phytoplankton. As we explored lower taxonomic ranks, we found inconsistent patterns at certain levels (e.g., phylum or order) within some taxa (e.g., Bacillariophyta and Rotifera), even substantial variability for several taxonomic groups (e.g., Euglenozoa and Apicomplexa). The varied environmental prevalence and persistence of eDNA, biological and ecological characteristics of diverse taxa in plankton, and sampling efficiency of both methods likely contributed to the observed disparities. Given its higher biodiversity recovery capacity, efficiency, sampling convenience, and capability to identify influential factors driving the geographical distribution of biodiversity, eDNA-based metabarcoding is recommended for studying plankton-environment interactions, particularly in studies involving a large number of samples at extended geographical scales.

Application of multiple binding sites for LAMP primers across P. falciparum genome improves detection of the parasite from whole blood samples

IntroductionMalaria remains a significant health concern, particularly in regions with widespread prevalence. As the transmission rates decrease, there is a rise in low-density infections with the causative parasite, P. falciparum, that often escape detection through standard point-of-care diagnostic tools. In-low transmission areas, even few undetected cases can trigger outbreaks, necessitating rapid and sensitive diagnostics. Loop-mediated isothermal Amplification (LAMP) stands out as a nucleic acid technique that can easily utilizes un-processed samples such of saliva, urine, and lysed whole blood templates for a sensitive detection. However, most nucleic acid tests detect genes with few copies per parasite making it difficult to detect low-density parasitaemia.MethodsWe selected Pfr364 multi-copy repeats of the P. falciparum genome as a target for amplification due to their higher copy number, ideal for rapid amplification, addressing amplification drawbacks of limited parasites DNA. We used a sequence clustering approach to design a novel set of LAMP primers, capable of binding to multiple sites. Subsequently, we developed a hydroxynaphthol blue (HNB) colorimetric LAMP assay, using genomic DNA obtained from the 3D7 strain cultivated in vitro. This assay’s performance was validated using archived clinical samples of both whole blood and matched saliva, ensuring accuracy through comparative analysis against gold standard, nested PCR, targeting the 18S RNA gene.ResultsThe HNB-LAMP assay achieved rapid amplification within 15 minutes and exhibited high sensitivity with a limit of detection of 1 parasite. Further, the LAMP assay was robust in whole blood lysed with Triton X-100 and heat-treated saliva clinical samples. Against nested PCR, the assay showed sensitivity of 100% for whole blood and 40% for saliva samples. Moreover, co-analysis with the nested PCR showed a perfect agreement between the two techniques. (K = 0.99 for whole blood, and 0.66 for saliva).ConclusionOur study presents a method for detecting P. falciparum using LAMP, which results in increased sensitivity, shorter assay times, and a simpler workflow than nucleic acid tests relying on conventional DNA extraction and additional equipment for result interpretation. These findings hold great promise for improved malaria diagnosis, especially in settings where low-density parasitaemia is prevalent and rapid and accurate malaria detection is crucial.

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

AbstractEsca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.

A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5'flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.

Regulation of DNA damage-induced HLA class I presentation

Immune checkpoint inhibitors (ICI) are cancer therapies that restore anti-tumor immunity; however, only a small percentage of patients have been completely cured by ICI alone. Multiple approaches in combination with other modalities have been used to improve the efficacy of ICI therapy. Among conventional cancer treatments, radiotherapy or DNA damage-based chemotherapy is a promising candidate as a partner of ICI because DNA damage signaling potentially stimulates immune activities turning the tumor’s immune environment into hot tumors. Programmed death-ligand 1 (PD-L1) and human leukocyte antigen class I (HLA-I), which are immune ligands, regulate the balance of anti-tumor immunity in the tumor microenvironment. PD-L1 functions as a brake to suppress cytotoxic T cell activity, whereas HLA-I is an immune accelerator that promotes the downstream of the T cell signaling. Accumulating evidence has demonstrated that DNA damage enhances the presentation of HLA-I on the surface of damaged cells. However, it is unclear how signal transduction in DNA-damaged cells upregulates the presentation of HLA-I with antigens. Our recent study uncovered the mechanism underlying DNA damage-induced HLA-I presentation, which requires polypeptide synthesis through a pioneer round of translation. In this review, we summarize the latest overview of how DNA damage stimulates antigen production presented by HLA-I.

Closing the Gap in Proteomic Identification of Histoplasma capsulatum: A Case Report and Review of Literature.

Histoplasmosis is a globally distributed systemic infection caused by the dimorphic fungus Histoplasma capsulatum (H. capsulatum). This fungus can cause a wide spectrum of clinical manifestations, and the diagnosis of progressive disseminated histoplasmosis is often a challenge for clinicians. Although microscopy and culture remain the gold standard diagnostic tests for Histoplasma identification, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has emerged as a method of microbial identification suitable for the confirmation of dimorphic fungi. However, to our knowledge, there are no entries for H. capsulatum spectra in most commercial databases. In this review, we describe the case of disseminated histoplasmosis in a patient living with HIV admitted to our university hospital that we failed to identify by the MALDI-TOF method due to the limited reference spectrum of the instrument database. Furthermore, we highlight the utility of molecular approaches, such as conventional polymerase chain reaction (PCR) and DNA sequencing, as alternative confirmatory tests to MALDI-TOF technology for identifying H. capsulatum from positive cultures. An overview of current evidence and limitations of MALDI-TOF-based characterization of H. capsulatum is also presented.

Fluorescent enzyme-based biosensor for sensitive analysis of DNA damage in cryopreserved sperm

The freeze-thaw process can induce irreversible structural and functional changes in human sperm, particularly sperm DNA damage. Selecting a more accurate and sensitive detection method for evaluating sperm DNA integrity is crucial. To accurately assess sperm DNA integrity following the freeze-thaw process and significantly improve the clinical and scientific utilization of cryopreserved sperm. In this study, we utilized a novel fluorescent biosensor, assisted by terminal deoxynucleotidyl transferase (TdT) and Endonuclease IV, to detect DNA breakpoints during sperm cryopreservation. We evaluated the biosensor's performance by comparing it with the conventional DNA fragmentation index (DFI) measured using sperm chromatin structure analysis (SCSA). The cryopreserved group exhibited a significantly higher sperm DFI compared to the fresh group. No significant difference was observed between the antioxidant group and the cryopreserved group. However, the new method revealed a significant reduction in the number of DNA breakpoints in the antioxidant group compared to the cryopreserved group. The novel biosensor demonstrated superior accuracy and effectiveness in assessing sperm DNA integrity during cryopreservation compared to the conventional SCSA method. We believe that the biosensor holds significant potential for widespread use in the field of reproductive medicine.

Preliminary study on prevalence of hemoprotozoan parasites harbored by Stomoxys (Diptera: Muscidae) and tabanid flies (Diptera: Tabanidae) in horse farms in Nakhon Si Thammarat province, Southern Thailand.

Stomoxys and tabanid flies are of medical and veterinary importance because they play crucial roles in disease transmission as mechanical vectors of various hemopathogens. However, its role as a hemoprotozoan parasite vector in horse farms has not been studied. Therefore, we investigated the occurrence of hemoprotozoan parasites belonging to the genera Babesia, Theileria, and Trypanosoma in Stomoxys and tabanid flies using conventional polymerase chain reaction (PCR) and DNA sequencing. All samples (Stomoxys and tabanid flies) were collected using an Nzi trap for three consecutive days each month from November 2022 to March 2023. The flies were morphologically identified to the species level and separated according to sex. Individual (for tabanid flies) or pooled samples (consisting of three specimens of Stomoxys flies of the same species and sex collected from the same site) were used for DNA extraction. Conventional PCR was used to screen for hemoprotozoan parasite DNA, followed by Sanger sequencing to identify the species. In total, 189 biting flies were collected, including four species of Stomoxys (Stomoxys bengalensis, Stomoxys calcitrans, Stomoxys indicus, and Stomoxys sitiens) and five species of tabanids (Atylotus cryptotaxis, Chrysops dispar, Tabanus megalops, Tabanus mesogaeus, and Tabanus rubidus). Stomoxys calcitrans was the most prevalent species, accounting for 58.7% (n = 111) of the collected flies. Ten (12.4%) of the 81 samples (individuals and pools) analyzed by PCR were positive for the 18S rRNA gene of the Theileria/Babesia species. Trypanosoma DNA was not detected in any sample. After performing Basic Local Alignment Search Tool searches and a phylogenetic analysis, only six samples (7.4%), including S. calcitrans (n = 2), S. sitiens (n = 2), T. megalops (n = 1), and A. cryptotaxis (n = 1), were found to be infected with Theileria sinensis. Furthermore, apicomplexan parasites, namely, Mattesia spp. and Colpodella spp., were found on S. indicus, the fungus Meira spp. was found on S. calcitrans, and the pathogenic green alga Helicosporidium spp. was found on A. cryptotaxis. This study is the first to report a variety of Stomoxys and tabanid flies collected from horse farms in Thailand, which were found to be infected with Theileria and Colpodella species that affect mammals, suggesting that Stomoxys and tabanid flies can be used to confirm the presence of hemoprotozoan parasites in the study area. Understanding the presence of hemoprotozoa in flies could help design vector control programs and manage various diseases in the study area.

"Total Human DNA Sampling" – Forensic DNA profiles from large areas

Employed for the first time in 1986, DNA profiling is nowadays established as one of the most widely used forensic techniques worldwide. However, until today, no efficient sampling technique existed to collect DNA from human skin cells from a large area, not to say from the floor of an entire room. This has been extremely unfortunate, as there is enormous forensic potential in these DNA traces from the ground to provide clues as to who has been present at a particular location, i.e. at the crime scene. By desquamation, humans loose several millions of skin cells per day, everywhere they stand, sit or walk; and they can do little about it. We developed a fast and simple method by which we can make use of all those lost skin cells. We use a vacuum cleaner equipped with a specialized filter cartridge to sample the ground. Fragmentation of the filter membrane and subsequent parallel processing of the filter fragments, using a modified Chelex® 100 extraction protocol, significantly reduce the complexity of the dust mixture. In this way, a large number of interpretable major contributor DNA profiles can be generated from individuals who have been present on the sampled surface. Overall, at least 38 % of the generated DNA profiles from all sampled test areas fulfilled the criteria for submission of single major contributor profiles to the Swiss DNA database. As demonstrated through a mock crime scene scenario simulating an indoor stabbing event, the perpetrator's DNA could be found on the floor even after a very short stay in the room of less than one minute. Furthermore, already the first application of the method at a real crime scene led to relevant case information for the police. Given its large investigative potential, we recommend Total Human DNA Sampling as a helpful complemental forensic tool to conventional DNA trace collection in major crimes.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Efficient Three-Dimensional DNA Nanomachine Guided by a Robust Tetrahedral DNA Nanoarray Structure for the Rapid and Ultrasensitive Electrochemical Detection of Matrix Metalloproteinase 2.

Herein, a giant-sized DNA nanoarray was subtly assembled by two kinds of independent tetrahedral DNA structures as the DNA track for a multi-armed three-dimensional (3D) DNA nanomachine to perform signal transduction and amplification efficiently, which was developed as an electrochemical biosensor for the rapid and ultrasensitive detection of matrix metalloproteinase 2 (MMP-2). Impressively, in contrast to conventional DNA walkers with inefficiency, which walked on random DNA tracks composed of a two-dimensional (2D) probe or a one-dimensional (1D) single-stranded (ss)DNA probe, the multi-armed 3D DNA nanomachine from exonuclease III (Exo III) enzyme-assisted target recycling amplification would be endowed with faster reaction speed and better walking efficiency because of the excellent rigidity and orderliness of the tetrahedral DNA nanoarray structure. Once the hairpin H3-label with the signal substance ferrocene (Fc) was added to the modified electrode surface, the multi-armed 3D DNA nanomachine would be driven to move along the well-designed nanoarray tracks by toehold-mediated DNA strand displacement, resulting in most of the ferrocene (Fc) binding to the electrode surface and a remarkable increase in electrochemical signals within 60 min. As a proof of concept, the prepared biosensor attained a low detection limit of 11.4 fg/mL for the sensitive detection of the target MMP-2 and was applied in Hela and MCF-7 cancer cell lysates. As a result, this strategy provided a high-performance sensing platform for protein detection in tumor diagnosis.

Target-Induced Stepwise Disintegration of Starlike Branched and Multiplex Embedded Systems for Amplified Detection of Serum MicroRNA.

DNA nanotechnology has shown great promise for biosensing and molecular recognition. However, the practical application of conventional DNA biosensors is constrained by inadequate target stimuli, intricate design schemes, multicomponent systems, and susceptibility to nuclease degradation. To overcome these limitations, we present a class of starlike branched and multiplex embedded system (SBES) with an integrated functional design and cascade exponential amplification for serum microRNA (miRNA) detection. The DNA arms can be integrated into an all-in-one system by surrounding a branch point, with each arm endowed with specific functionalities by embedding different DNA fragments. These fragments include a segment complementary to the target miRNA for the recognition element, palindromic tails for self-primed polymerization, and a region with the same sequences as the target serving as the target analogue. Upon exposure to a target miRNA, the DNA arms unwind in a stepwise manner through palindrome-mediated dimerization and polymerization. This enables target recycling for subsequent reactions while releasing the target analogue to generate a secondary response in a feedback manner. A comparative analysis illustrates that the signal-to-noise ratio (SNR) of a full SBES with a feedback strategy is approximately 250% higher than the system without a feedback design. We demonstrate that the four-arm 4pSBES has the benefits of multifunctional integration, enhanced sensitivity, and low false-positive signals, which makes this approach ideally suited for clinical diagnosis. Moreover, an upgraded SBES with additional DNA arms (e.g., 6pSBES) can be constructed to allow multifunctional extension, offering unprecedented opportunities to build versatile DNA nanostructures for biosensing.

NuMY-A qPCR Assay Simultaneously Targeting Human Autosomal, Y-Chromosomal, and Mitochondrial DNA.

The accurate quantification of DNA in forensic samples is of utmost importance. These samples are often present in limited amounts; therefore, it is indicated to use the appropriate analysis route with the optimum DNA amount (when possible). Also, DNA quantification can inform about the degradation stage and therefore support the decision on which downstream genotyping method to use. Consequently, DNA quantification aids in getting the best possible results from a forensic sample, considering both its DNA quantity and quality limitations. Here, we introduce NuMY, a new quantitative real-time PCR (qPCR) method for the parallel quantification of human nuclear (n) and mitochondrial (mt) DNA, assessing the male portion in mixtures of both sexes and testing for possible PCR inhibition. NuMY is based on previous work and follows the MIQE guidelines whenever applicable. Although quantification of nuclear (n)DNA by simultaneously analyzing autosomal and male-specific targets is available in commercial qPCR kits, tools that include the quantification of mtDNA are sparse. The quantification of mtDNA has proven relevant for samples with low nDNA content when conventional DNA fingerprinting techniques cannot be followed. Furthermore, the development and use of new massively parallel sequencing assays that combine multiple marker types, i.e., autosomal, Y-chromosomal, and mtDNA, can be optimized when precisely knowing the amount of each DNA component present in the input sample. For high-quality DNA extracts, NuMY provided nDNA results comparable to those of another quantification technique and has also proven to be a reliable tool for challenging, forensically relevant samples such as mixtures, inhibited, and naturally degraded samples.