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The Journal of Molecular Diagnostics :... Oct 2020DNA copy number alterations (CNAs) are promising biomarkers to predict prostate cancer (PCa) outcome. However, fluorescence in situ hybridization (FISH) cannot assess...
Design and Development of a Fully Synthetic Multiplex Ligation-Dependent Probe Amplification-Based Probe Mix for Detection of Copy Number Alterations in Prostate Cancer Formalin-Fixed, Paraffin-Embedded Tissue Samples.
DNA copy number alterations (CNAs) are promising biomarkers to predict prostate cancer (PCa) outcome. However, fluorescence in situ hybridization (FISH) cannot assess complex CNA signatures because of low multiplexing capabilities. Multiplex ligation-dependent probe amplification (MLPA) can detect multiple CNAs in a single PCR assay, but PCa-specific probe mixes available commercially are lacking. Synthetic MLPA probes were designed to target 10 CNAs relevant to PCa: 5q15-21.1 (CHD1), 6q15 (MAP3K7), 8p21.2 (NKX3-1), 8q24.21 (MYC), 10q23.31 (PTEN), 12p13.1 (CDKN1B), 13q14.2 (RB1), 16p13.3 (PDPK1), 16q23.1 (GABARAPL2), and 17p13.1 (TP53), with 9 control probes. In cell lines, CNAs were detected when the cancer genome was as low as 30%. Compared with FISH in radical prostatectomy formalin-fixed, paraffin-embedded samples (n = 18: 15 cancers and 3 matched benign), the MLPA assay showed median sensitivity and specificity of 80% and 93%, respectively, across all CNAs assessed. In the validation set (n = 40: 20 tumors sampled in two areas), the respective sensitivity and specificity of MLPA compared advantageously with FISH and TaqMan droplet digital PCR (ddPCR) when assessing PTEN deletion (FISH: 85% and 100%; ddPCR: 100% and 83%) and PDPK1 gain (FISH: 100% and 92%; ddPCR: 93% and 100%). This new PCa probe mix accurately identifies CNAs by MLPA across multiple genes using low quality and quantities (50 ng) of DNA extracted from clinical formalin-fixed, paraffin-embedded samples.
Topics: Cell Line, Tumor; DNA Copy Number Variations; DNA Probes; DNA, Neoplasm; Formaldehyde; Genome, Human; Humans; Limit of Detection; Male; Nucleic Acid Amplification Techniques; Paraffin Embedding; Prostatic Neoplasms; Reproducibility of Results; Tissue Fixation
PubMed: 32763409
DOI: 10.1016/j.jmoldx.2020.07.003 -
Nucleic Acids Research Jul 2016We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method...
We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.
Topics: Animals; DNA Ligases; DNA Probes; Humans; Mice; MicroRNAs; Nucleic Acid Hybridization; Rats; Real-Time Polymerase Chain Reaction; Viral Proteins
PubMed: 27154271
DOI: 10.1093/nar/gkw399 -
Cancer Genomics & Proteomics 2023Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors...
BACKGROUND/AIM
Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed.
MATERIALS AND METHODS
Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations.
RESULTS
The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein.
CONCLUSION
PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.
Topics: Animals; Humans; Mice; Carcinoma, Non-Small-Cell Lung; DNA; DNA Probes; ErbB Receptors; Lung Neoplasms; Mice, Nude; Mutation; Peptide Nucleic Acids; Protein Kinase Inhibitors
PubMed: 37400147
DOI: 10.21873/cgp.20389 -
Biosensors & Bioelectronics Jan 2017This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the...
This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the effect of contaminants in commercial thiolated DNA probe, the electrode surface was functionalized with the 24-nucleotide DNA probes based on the West Nile virus sequence (Kunjin strain). The biosensor has the ability to detect complementary DNA fragments with a detection limit down to 20 DNA target molecules (1.5aM range), making it suitable for a practical point-of-care (POC) platform for low target count clinical applications without the need for amplification. The reproducibility of the biosensor detection was improved with efficient covalent immobilization of purified single-stranded DNA probe oligomers on cleaned gold microelectrodes. In addition to the low detection limit, the biosensor showed a dynamic range of detection from 1µL to 10µL target molecules (20 to 2 million targets), making it suitable for sample analysis in a typical clinical application environment. The binding results presented in this paper were validated using fluorescent oligomers.
Topics: Base Sequence; Biosensing Techniques; DNA; DNA Probes; DNA, Single-Stranded; Electric Capacitance; Electrochemical Techniques; Electrodes; Equipment Design; Gold; Humans; Immobilized Nucleic Acids; Limit of Detection; Nucleic Acid Hybridization; Point-of-Care Systems; Reproducibility of Results; Sulfhydryl Compounds; West Nile Fever; West Nile virus
PubMed: 27619528
DOI: 10.1016/j.bios.2016.09.006 -
Scientific Reports Jul 2020Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic...
Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA-DNA duplexes formation, by recording ionic current blockades signature of AuNP-α-HL interactions. This approach offers advantages including: (1) a simple to operate platform, producing clear-cut readout signals based on distinct size differences of PNA-induced AuNPs aggregates, in relation to the presence in solution of complementary ssDNAs to the PNA fragments (2) sensitive and selective detection of target ssDNAs (3) specific ssDNA detection in the presence of interference DNA, without sample labeling or signal amplification. The powerful synergy of protein nanopore-based nanoparticle detection and specific PNA-DNA hybridization introduces a new strategy for nucleic acids biosensing with short detection time and label-free operation.
Topics: Biosensing Techniques; DNA Probes; DNA, Single-Stranded; Gold; Hemolysin Proteins; Metal Nanoparticles; Nanopores; Nucleic Acid Hybridization; Peptide Nucleic Acids
PubMed: 32647249
DOI: 10.1038/s41598-020-68258-x -
Biosensors Mar 2024Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges...
Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges arise in protein microarrays due to the heterogeneous nature of proteins and, thus, differences in their immobilization conditions. This article advocates DNA-directed immobilization (DDI) as a solution, emphasizing its rapid and cost-effective fabrication of biosensing platforms. Thiolated single-stranded DNA and its analogues, such as ZNA and PNA probes, were used to immobilize model proteins (-CRP antibodies and SARS-CoV nucleoprotein). The study explores factors influencing DDI-based immunosensor performance, including the purity of protein-DNA conjugates and the stability of their duplexes with DNA and analogues. It also provides insight into backfilling agent type and probe surface density. The research reveals that single-component monolayers lack protection against protein adsorption, while mixing the probes with long-chain ligands may hinder DNA-protein conjugate anchoring. Conventional DNA probes offer slightly higher surface density, while ZNA probes exhibit better binding efficiency. Despite no enhanced stability in different ionic strength media, the cost-effectiveness of DNA probes led to their preference. The findings contribute to advancing microarray technology, paving the way for new generations of DDI-based multiplex platforms for rapid and robust diagnostics.
Topics: Biosensing Techniques; Immunoassay; DNA; DNA Probes; Proteins; Antigens; Biology
PubMed: 38534243
DOI: 10.3390/bios14030136 -
Proceedings of the National Academy of... Apr 2020Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In...
Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target-probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.
Topics: Computational Biology; Computer Simulation; Computer-Aided Design; DNA Probes; DNA, Bacterial; Genome, Bacterial; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 32241887
DOI: 10.1073/pnas.1918274117 -
Biosensors Jul 2022MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system...
MicroRNAs play a vital role in cancer development and are considered as potential biomarkers for early prognostic assessment. Here, we propose a novel biosensing system to achieve fluorescence imaging of miRNA21 (miR21) in cancer cells. This system consists of two components: an optimized "off-on" double-stranded DNA (dsDNA) fluorescent for miR21 sensing by efficient strand-displacement reaction and a potent carrier vesicle, termed niosome (SPN), to facilitate the efficient intracellular delivery of the dsDNA probe. A series of dsDNA probes based on fluorescence energy resonance transfer (FRET) was assembled to target miR21. By optimizing the appropriate length of the reporter strand in the dsDNA probe, high accuracy and sensitivity for miR21 recognition are ensured. To overcome the cellular barrier, we synthesized SPN with the main components of a nonionic surfactant Span 80 and a cationic lipid DOTAP, which could efficiently load dsDNA probes via electrostatic interactions and potently deliver the dsDNA probes into cells with good biosafety. The SPN/dsDNA achieved efficient miR21 fluorescent imaging in living cells, and could discriminate cancer cells (MCF-7) from normal cells (L-02). Therefore, the proposed SPN/dsDNA system provides a powerful tool for intracellular miRNA biosensing, which holds great promise for early cancer diagnosis.
Topics: Biosensing Techniques; DNA; DNA Probes; Fluorescent Dyes; Liposomes; Optical Imaging
PubMed: 35892454
DOI: 10.3390/bios12080557 -
Nanoscale Jan 2019The development of ultrasensitive methods for detecting specific genes and discriminating single nucleotide polymorphisms (SNPs) is important for biomedical research and...
The development of ultrasensitive methods for detecting specific genes and discriminating single nucleotide polymorphisms (SNPs) is important for biomedical research and clinical disease diagnosis. Herein, we report an ultrasensitive approach for label-free detection and discrimination of a full-match target-DNA from its cancer related SNPs by combining magnetic nanoparticle (MNP) capture and poly-enzyme nanobead signal amplification. It uses a MNP linked capture-DNA and a biotinylated signal-DNA to sandwich the target followed by ligation to offer high SNP discrimination: only the perfect-match target-DNA yields a covalently linked biotinylated signal-DNA on the MNP surface for subsequent binding to a neutravidin-horseradish peroxidase conjugate (NAV-HRP) for signal amplification. The use of polymer nanobeads each tagged with thousands of copies of HRPs greatly improves the signal amplification power, allowing for direct, amplification-free quantification of low aM target-DNA over 6 orders of magnitude (0.001-1000 fM). Moreover, this sensor also offers excellent discrimination between the perfect-match gene and its cancer-related SNPs and can positively detect 1 fM perfect-match target-DNA in the presence of 100 fold excess of co-existing single-base mismatch targets. Furthermore, it works robustly in clinically relevant media (e.g. 10% human serum) and gives even higher SNP discrimination than that in clean buffers. This ultrasensitive DNA sensor appears to have excellent potential for rapid detection and diagnosis of genetic diseases.
Topics: Avidin; Base Pair Mismatch; Biosensing Techniques; Biotinylation; Click Chemistry; DNA; DNA Probes; Horseradish Peroxidase; Limit of Detection; Magnetite Nanoparticles; Polyethylene Glycols; Polymorphism, Single Nucleotide
PubMed: 30601516
DOI: 10.1039/c8nr07641c -
PloS One 2015Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction...
Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.
Topics: Animals; Base Sequence; Circovirus; DNA Probes; Limit of Detection; Nanoparticles; Polymerase Chain Reaction; Reproducibility of Results; Swine; Swine Diseases; Transmissible gastroenteritis virus
PubMed: 26544710
DOI: 10.1371/journal.pone.0141545