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Scientific Reports Jul 2023The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination...
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA's integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.
Topics: DNA, Complementary; Reverse Transcription; Polymerase Chain Reaction; DNA; RNA; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37454173
DOI: 10.1038/s41598-023-38383-4 -
ACS Nano Apr 2024DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of...
DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a roximity activation mechanism that converts protein input into DNA barcodes for the DNA xponential mplification eaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.
Topics: DNA; Nucleic Acid Amplification Techniques; Biosensing Techniques
PubMed: 38572806
DOI: 10.1021/acsnano.3c11435 -
Analytical Chemistry Jan 2024Fluorescence hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids...
Fluorescence hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.
Topics: Humans; In Situ Hybridization, Fluorescence; Lanthanoid Series Elements; HeLa Cells; RNA; DNA; DNA Probes
PubMed: 38214216
DOI: 10.1021/acs.analchem.3c04274 -
Nucleic Acids Research Sep 2023The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A...
The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.
Topics: Humans; COVID-19; DNA; Mutation; SARS-CoV-2; Polymorphism, Genetic; RNA; Genetic Techniques
PubMed: 37562941
DOI: 10.1093/nar/gkad601 -
Nanoscale Sep 2023Identification of non-amplified DNA sequences and single-base mutations is essential for molecular biology and genetic diagnostics. This paper reports a novel sensor...
Identification of non-amplified DNA sequences and single-base mutations is essential for molecular biology and genetic diagnostics. This paper reports a novel sensor consisting of electrochemically-gated graphene coplanar waveguides coupled with a microfluidic channel. Upon exposure to analytes, propagation of electromagnetic waves in the waveguides is modified as a result of interactions with the fringing field and modulation of graphene dynamic conductivity resulting from electrostatic gating. Probe DNA sequences are immobilised on the graphene surface, and the sensor is exposed to DNA sequences which either perfectly match the probe, contain a single-base mismatch or are unrelated. By monitoring the scattering parameters at frequencies between 50 MHz and 50 GHz, unambiguous and reproducible discrimination of the different strands is achieved at concentrations as low as one attomole per litre (1 aM). By controlling and synchronising frequency sweeps, electrochemical gating, and liquid flow in the microfluidic channel, the sensor generates multidimensional datasets. Advanced data analysis techniques are utilised to take full advantage of the richness of the dataset. A classification accuracy >97% between all three sequences is achieved using different Machine Learning models, even in the presence of simulated noise and low signal-to-noise ratios. The sensor exceeds state-of-the-art sensitivity of field-effect transistors and microwave sensors for the identification of single-base mismatches.
Topics: Graphite; Microwaves; Biosensing Techniques; DNA Probes; Signal-To-Noise Ratio
PubMed: 37682040
DOI: 10.1039/d3nr01239e -
Advanced Materials (Deerfield Beach,... Nov 2023Ultrasensitive identification of biomarkers in biofluids is essential for the precise diagnosis of diseases. For the gold standard approaches, polymerase chain reaction...
Ultrasensitive identification of biomarkers in biofluids is essential for the precise diagnosis of diseases. For the gold standard approaches, polymerase chain reaction and enzyme-linked immunosorbent assay, cumbersome operational steps hinder their point-of-care applications. Here, a bionic biomarker entrapment system (BioES) is implemented, which employs a multi-body Y-shaped tetrahedral DNA probe immobilized on carbon nanotube transistors. Clinical identification of endometriosis is successfully realized by detecting an estrogen receptor, ERβ, from the lesion tissue of endometriosis patients and establishing a standard diagnosis procedure. The multi-body Y-shaped BioES achieves a theoretical limit of detection (LoD) of 6.74 aM and a limit of quantification of 141 aM in a complex protein milieu. Furthermore, the BioES is optimized into a multi-site recognition module for enhanced binding efficiency, realizing the first identification of monkeypox virus antigen A35R and unamplified detection of circulating tumor DNA of breast cancer in serum. The rigid and compact probe framework with synergy effect enables the BioES to target A35R and DNA with a LoD down to 991 and 0.21 aM, respectively. Owing to its versatility for proteins and nucleic acids as well as ease of manipulation and ultra-sensitivity, the BioES can be leveraged as an all-encompassing tool for population-wide screening of epidemics and clinical disease diagnosis.
Topics: Female; Humans; Endometriosis; Biomarkers; DNA; DNA Probes; Nanotubes, Carbon; Biosensing Techniques; Limit of Detection
PubMed: 37486783
DOI: 10.1002/adma.202304119 -
Molecular Phylogenetics and Evolution Oct 2023Sequence capture of ultraconserved elements (UCEs) has transformed molecular systematics across many taxa, with arachnids being no exception. The probe set available for...
Sequence capture of ultraconserved elements (UCEs) has transformed molecular systematics across many taxa, with arachnids being no exception. The probe set available for Arachnida has been repeatedly used across multiple arachnid lineages and taxonomic levels, however more specific probe sets for spiders have demonstrated that more UCEs can be recovered with higher probe specificity. In this study, we develop an Opiliones-specific UCE probe set targeting 1915 UCEs using a combination of probes designed from genomes and transcriptomes, as well as the most useful probes from the Arachnida probe set. We demonstrate the effectiveness of this probe set across Opiliones with the most complete family-level phylogeny made to date, including representatives from 61 of 63 currently described families. We also test UCE recovery from historical specimens with degraded DNA, examine population-level data sets, and assess "backwards compatibility" with samples hybridized with the Arachnida probe set. The resulting phylogenies - which include specimens hybridized using both the Opiliones and Arachnida probe sets, historical specimens, and transcriptomes - are largely congruent with previous multi-locus and phylogenomic analyses. The probe set is also "backwards compatible", increasing the number of loci obtained in samples previously hybridized with the Arachnida probe set, and shows high utility down to shallow population-level divergences. This probe set has the potential to further transform Opiliones molecular systematics, resolving many long-standing taxonomic issues plaguing this lineage.
PubMed: 37479049
DOI: 10.1016/j.ympev.2023.107887 -
PloS One 2023Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other...
Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease.
Topics: Infant, Newborn; Humans; Cell-Free Nucleic Acids; DNA; Nucleic Acids; Persistent Infection; Chagas Disease; DNA Primers; Extracellular Vesicles; Neglected Diseases
PubMed: 37682970
DOI: 10.1371/journal.pone.0282814 -
Journal of Biotechnology Feb 2024Photoacoustic imaging(PAI) is a widely developing imaging modality that has seen tremendous evolvement in the last decade. PAI has gained the upper hand in the imaging... (Review)
Review
Photoacoustic imaging(PAI) is a widely developing imaging modality that has seen tremendous evolvement in the last decade. PAI has gained the upper hand in the imaging field as it takes advantage of optical absorption and ultrasound detection that imparts higher resolution, rich contrast and elevated penetration depth. Unlike other imaging techniques, PAI does not use ionising radiation and is a better, cost-effective and healthier alternative to other imaging techniques. It offers greater specificity than conventional ultrasound imaging with the ability to detect haemoglobin, lipids, water and other light-absorbing chromophores. These properties of PAI have led to its extended applications in the biomedical field in the treatment of diseases such as cancer. This paper reviews how DNA probes have been used in PAI, the various techniques by which it has been modified, and their role in the process. We also focus on different nanocomposites containing DNA having PAI and photothermal therapy(PTT) properties for detection, diagnosis and therapy, its constituents and the role of DNA in it.
Topics: Humans; Photoacoustic Techniques; Phototherapy; Neoplasms; DNA Probes
PubMed: 38211667
DOI: 10.1016/j.jbiotec.2023.12.019 -
Bioelectrochemistry (Amsterdam,... Oct 2023Electrochemical aptasensor has been broadly advanced for nucleic acid detection. However, it is a long-term goal to design an aptasensor with high specificity,...
Electrochemical aptasensor has been broadly advanced for nucleic acid detection. However, it is a long-term goal to design an aptasensor with high specificity, flexibility, and simplicity. In this work, we develop a strategy of triblock DNA probe, which consists of two DNA probes at both ends and ployA fragments in the middle as probe-polyA-probe. PolyA fragment has high affinity to the surface of gold electrode, so it can be assembled on the electrode surface via polyA instead of traditional Au-S bonds. When the target DNA is simultaneously hybridized with the two capture probes, the hybridization stability can be improved due to the strong base stacking effect. [Ru(NH)], as signal probe, can be electrostatically adsorbed on the negatively charged DNA skeleton. A wide linear range (10 pM-10 μM) is obtained with a detection limit of 2.9 pM. Our electrochemical aptasensor has good repeatability, stability, and specificity. More importantly, the electrochemical sensor can successfully detect DNA in human serum samples, which proves its practical value and extensive applicability in complex environment.
Topics: Humans; Electrochemical Techniques; Limit of Detection; Aptamers, Nucleotide; DNA; Biosensing Techniques; Gold
PubMed: 37379739
DOI: 10.1016/j.bioelechem.2023.108494