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Journal of Insect Science (Online) Sep 2023The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from...
The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.
Topics: Animals; Culicidae; Culex; DNA; Polymerase Chain Reaction; DNA Primers
PubMed: 37804500
DOI: 10.1093/jisesa/iead080 -
The Analyst Dec 2023The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from...
The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from time-consuming operations and/or a narrow detection range. In this work, for the rapid determination of bevacizumab in plasma, a series of chimeric hairpin DNA aptamer-based probes were designed by the modification, labeling and theoretical computation of an original aptamer. Then, the dissociation constant of the modified hairpin DNA to bevacizumab was measured and screened using microscale thermophoresis. The best chimeric hairpin DNA aptamer-based probe was then selected, and a one-step platform for the rapid determination of bevacizumab was constructed. This strategy has the advantages of being simple, fast and label-free. Because of the design and screening of the hairpin DNA, as well as the optimization of the concentration and electrochemical parameters, a low detection limit of 0.37 pM (0.054 ng mL) with a wide linear range (1 pM-1 μM) was obtained. Finally, the rationally constructed biosensor was successfully applied to the determination of bevacizumab in spiked samples, and it showed good accuracy and precision. This method is expected to truly realize accurate and rapid detection of bevacizumab and provides a new idea for the precise treatment of diseases.
Topics: Aptamers, Nucleotide; Bevacizumab; Biosensing Techniques; DNA; DNA Probes; Limit of Detection; Electrochemical Techniques
PubMed: 38018757
DOI: 10.1039/d3an01324c -
Journal of Materials Chemistry. B Sep 2023Plasmonic gold nanorod (AuNR) on a macromolecular matrix exhibits an end-to-end (ETE) long-range self-assembly (AuNR) with > 100. In the case of small molecules as a...
Plasmonic gold nanorod (AuNR) on a macromolecular matrix exhibits an end-to-end (ETE) long-range self-assembly (AuNR) with > 100. In the case of small molecules as a template, the pre-synthesized macromolecular matrix is missing and this brings a synthetic challenge in directed long-range assembly of AuNR. Self-assembly with thiol-modified small DNA and AuNR shows a much short-range ETE assembly with < 25 a simple evaporation technique on a solid surface. In this study, the introduction of two short amine modified probe DNAs (∼2.5 nm) and one 22-mer complementary single strand (ss)-DNA template (∼7 nm) show the long-range ETE self-assembly of (AuNR) with > 130. In the solution state, the zigzag arrangement within the assembled structure controls the typical change in the absorption behavior for (AuNR) ETE assembly. The formation of this long-range ETE self-assembly in a solution state was verified from the combined effect of fluorescence resonance energy transfer (FRET) and hotspot-induced fluorescence enhancement. The probe DNAs and templated DNA concentration on fluorescence enhancement have been varied to monitor the effect of (AuNR) with = ∼5-130 in ETE self-assembly. Primarily quenched FRET acceptor in the presence of AuNR decisively exhibits remarkable fluorogenic response in ETE self-assembly with maximum value. Although the FRET efficiencies among the fluorophores are comparable, the fluorogenic boost in ETE AuNR is due to the increased number of intrinsic navigated hotspots in these assemblies.
Topics: DNA; DNA, Complementary; DNA, Single-Stranded; Gold; Nanotubes
PubMed: 37721049
DOI: 10.1039/d3tb01446k -
Talanta Nov 2023Cardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and...
Cardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015-1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.
Topics: Humans; Warfarin; Polymorphism, Single Nucleotide; Pharmacogenetics; Cytochrome P-450 CYP2C9; Aryl Hydrocarbon Hydroxylases; Vitamin K Epoxide Reductases; Anticoagulants; DNA; Genotype; DNA Probes; Biosensing Techniques
PubMed: 37276677
DOI: 10.1016/j.talanta.2023.124692 -
Archives of Razi Institute Aug 2023Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the...
Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.
Topics: Animals; Bombyx; Microsporidiosis; Real-Time Polymerase Chain Reaction; DNA Primers; DNA
PubMed: 38226388
DOI: 10.32592/ARI.2023.78.4.1185 -
Briefings in Bioinformatics Jan 2024Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However,...
Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However, their success depends on the performance of probes and the evenness of sequencing depth among each probe. To accurately predict probe coverage depth, a model called Deqformer is proposed in this study. Deqformer utilizes the oligonucleotides sequence of each probe, drawing inspiration from Watson-Crick base pairing and incorporating two BERT encoders to capture the underlying information from the forward and reverse probe strands, respectively. The encoded data are combined with a feed-forward network to make precise predictions of sequencing depth. The performance of Deqformer is evaluated on four different datasets: SNP panel with 38 200 probes, lncRNA panel with 2000 probes, synthetic panel with 5899 probes and HD-Marker panel for Yesso scallop with 11 000 probes. The SNP and synthetic panels achieve impressive factor 3 of accuracy (F3acc) of 96.24% and 99.66% in 5-fold cross-validation. F3acc rates of over 87.33% and 72.56% are obtained when training on the SNP panel and evaluating performance on the lncRNA and HD-Marker datasets, respectively. Our analysis reveals that Deqformer effectively captures hybridization patterns, making it robust for accurate predictions in various scenarios. Deqformer leads to a novel perspective for probe design pipeline, aiming to enhance efficiency and effectiveness in probe design tasks.
Topics: DNA Probes; Deep Learning; RNA, Long Noncoding; Nucleic Acid Hybridization; Genomics
PubMed: 38305453
DOI: 10.1093/bib/bbae007 -
Applied Physics Reviews Mar 2024Graphene-based materials and DNA probes/nanostructures have emerged as building blocks for constructing powerful biosensors. Graphene-based materials possess exceptional...
Graphene-based materials and DNA probes/nanostructures have emerged as building blocks for constructing powerful biosensors. Graphene-based materials possess exceptional properties, including two-dimensional atomically flat basal planes for biomolecule binding. DNA probes serve as excellent selective probes, exhibiting specific recognition capabilities toward diverse target analytes. Meanwhile, DNA nanostructures function as placement scaffolds, enabling the precise organization of molecular species at nanoscale and the positioning of complex biomolecular assays. The interplay of DNA probes/nanostructures and graphene-based materials has fostered the creation of intricate hybrid materials with user-defined architectures. This advancement has resulted in significant progress in developing novel biosensors for detecting DNA, RNA, small molecules, and proteins, as well as for DNA sequencing. Consequently, a profound understanding of the interactions between DNA and graphene-based materials is key to developing these biological devices. In this review, we systematically discussed the current comprehension of the interaction between DNA probes and graphene-based materials, and elucidated the latest advancements in DNA probe-graphene-based biosensors. Additionally, we concisely summarized recent research endeavors involving the deposition of DNA nanostructures on graphene-based materials and explored imminent biosensing applications by seamlessly integrating DNA nanostructures with graphene-based materials. Finally, we delineated the primary challenges and provided prospective insights into this rapidly developing field. We envision that this review will aid researchers in understanding the interactions between DNA and graphene-based materials, gaining deeper insight into the biosensing mechanisms of DNA-graphene-based biosensors, and designing novel biosensors for desired applications.
PubMed: 38784221
DOI: 10.1063/5.0171364 -
Analytical Chemistry Aug 2023Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and...
Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.
Topics: Humans; Mutation; Polymerase Chain Reaction; DNA; Lung Neoplasms; DNA Primers
PubMed: 37527514
DOI: 10.1021/acs.analchem.3c01803 -
BMC Biotechnology Aug 2023Global efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health...
BACKGROUND
Global efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).
RESULTS
Our approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.
CONCLUSIONS
Overall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.
Topics: Humans; Communicable Diseases; High-Throughput Nucleotide Sequencing; Biological Assay; Computational Biology; Gene Library
PubMed: 37612665
DOI: 10.1186/s12896-023-00804-7 -
PLoS Computational Biology Oct 2023Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet...
Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read sequencing libraries lack depth and strand-of-origin information in cDNA-based methods, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to "strand" long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we also present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5' and 3' ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.
Topics: DNA, Complementary; RNA, Long Noncoding; Sequence Analysis, RNA; Transcriptome; Gene Library; Protein Isoforms; High-Throughput Nucleotide Sequencing
PubMed: 37883581
DOI: 10.1371/journal.pcbi.1011576