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ACS Sensors Apr 2022MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked...
MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked miRNAs is vital to the emerging revolution in early diagnosis of diseases. While the detection of miRNAs is a challenge due to their intrinsic properties such as small size, high sequence similarity among miRNAs and low abundance in biological fluids, the majority of miRNA-detection strategies involve either target/signal amplification or involve complex sensing designs. In this study, we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) expressed by triple-negative breast cancer (TNBC) cells. The sensor shows a relatively low FRET state in the absence of a target but it undergoes continuous FRET transitions between low- and high-FRET states in the presence of the target. The sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300 000 fM. Using this strategy, we demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts. Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.
Topics: DNA Probes; Fluorescence Resonance Energy Transfer; Humans; MicroRNAs; Nucleic Acid Hybridization; Triple Negative Breast Neoplasms
PubMed: 35312280
DOI: 10.1021/acssensors.1c02748 -
Biosensors Jul 2022DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect...
DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBR Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5-110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.
Topics: Biosensing Techniques; DNA; DNA Primers; Limit of Detection; Methyltransferases; Nucleic Acid Amplification Techniques
PubMed: 35892466
DOI: 10.3390/bios12080569 -
Molecules (Basel, Switzerland) May 2023Honey is a widely consumed natural product, and its entomological origin can significantly influence its market value. Therefore, traceability of the entomological... (Review)
Review
Honey is a widely consumed natural product, and its entomological origin can significantly influence its market value. Therefore, traceability of the entomological origin of honey should also be considered in honey quality control protocols. Although several methods exist, such as physicochemical characterization and bioactivity profiling of honey of different entomological origins, the most promising three methods for entomological authentication of honey include protein-based identification, chemical profiling, and a DNA-based method. All of these methods can be applied for reliable identification of the entomological origin of honey. However, as the honey is a complex matrix, the inconsistency of the results obtained by these methods is a pragmatic challenge, and therefore, the use of each method in all the cases is questionable. Most of these methodologies can be used for authentication of newly harvested honey and it is worth understanding the possibility of using these methods for authentication of relatively old samples. Most probably, using DNA-based methods targeting small fragments of DNA can provide the best result in old samples, however, the species-specific primers targeting short fragments are limited and not available for all species. Therefore, using universal primers in combination with a DNA metabarcoding approach can be a good solution that requires further investigation. This present article describes the applications of different methods, their pros, and their cons to identify honey based on entomological origin.
Topics: Honey; DNA; DNA Primers; Species Specificity; Biological Products
PubMed: 37241972
DOI: 10.3390/molecules28104232 -
Sensors (Basel, Switzerland) Mar 2021The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich....
The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse's feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria and have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse's diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of capture probe 1 (SECP1) and target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.
Topics: Acidosis; Animals; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Firmicutes; Gold; Horses; Nucleic Acid Hybridization; Streptococcus bovis
PubMed: 33810389
DOI: 10.3390/s21072319 -
ACS Sensors Mar 2021Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of...
Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of diagnostic methods capable of detecting diseases at the molecular level in biological fluids is vital to the emerging revolution in the early diagnosis of diseases. However, the vast majority of the currently available ultrasensitive detection strategies involve either target/signal amplification or involve complex designs. Here, using a p53 tumor suppressor gene whose mutation has been implicated in more than 50% of human cancers, we show a background-free ultrasensitive detection of this gene on a simple platform. The sensor exhibits a relatively static mid-FRET state in the absence of a target that can be attributed to the time-averaged fluorescence intensity of fast transitions among multiple states, but it undergoes continuous dynamic switching between a low- and a high-FRET state in the presence of a target, allowing a high-confidence detection. In addition to its simple design, the sensor has a detection limit down to low femtomolar (fM) concentration without the need for target amplification. We also show that this sensor is highly effective in discriminating against single-nucleotide polymorphisms (SNPs). Given the generic hybridization-based detection platform, the sensing strategy developed here can be used to detect a wide range of nucleic acid sequences enabling early diagnosis of diseases and screening genetic disorders.
Topics: Base Sequence; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Humans; Nucleic Acid Hybridization
PubMed: 33720708
DOI: 10.1021/acssensors.1c00002 -
Molecules (Basel, Switzerland) Dec 2022Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better...
Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.
Topics: CRISPR-Cas Systems; DNA; Polynucleotide 5'-Hydroxyl-Kinase; DNA Probes; Fluorescent Dyes; RNA
PubMed: 36558152
DOI: 10.3390/molecules27249019 -
International Journal of Molecular... Jul 2021Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome... (Review)
Review
Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.
Topics: Chromosomes, Plant; DNA Probes; Fluorescence; Genome; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Oligonucleotides; Repetitive Sequences, Nucleic Acid; Research Design; Sequence Analysis, DNA
PubMed: 34281175
DOI: 10.3390/ijms22137124 -
Current Protocols Oct 2022This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled...
This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.
Topics: DNA, Complementary; Trypan Blue; Sequence Analysis, RNA; Sucrose; Oligonucleotides; Lipids
PubMed: 36286606
DOI: 10.1002/cpz1.579 -
Organic & Biomolecular Chemistry Nov 2022Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and...
Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), , single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with ()-hydroxymethyl moieties, (ii) Invader probes, , DNA duplexes modified with +1 interstrand zippers of 2'--(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, , duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.
Topics: Serine; In Situ Hybridization, Fluorescence; DNA; Peptide Nucleic Acids; RNA; DNA Probes
PubMed: 36285843
DOI: 10.1039/d2ob01567f -
BMC Infectious Diseases Jan 2020Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance.... (Review)
Review
BACKGROUND
Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507-511), Probe B (codons 512-518), Probe C (codons 518-523), Probe D (codons 523-529) and Probe E (codons 529-533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results.
METHODS
Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample.
RESULTS
A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations.
CONCLUSION
Mutations associated with Probe E (codons 529-533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Codon; DNA; Drug Resistance, Bacterial; Ethiopia; Female; Genetic Testing; Humans; Infant; Male; Middle Aged; Mutation; Mycobacterium tuberculosis; Retrospective Studies; Rifampin; Sensitivity and Specificity; Sputum; Tuberculosis; Young Adult
PubMed: 32000702
DOI: 10.1186/s12879-020-4817-2