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Scientific Reports Sep 2019Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of...
Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.
Topics: Costs and Cost Analysis; DNA Probes; DNA, Single-Stranded; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Thrombin
PubMed: 31558745
DOI: 10.1038/s41598-019-50371-1 -
Molecules (Basel, Switzerland) Dec 2022The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an...
The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman's rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.
Topics: In Situ Hybridization, Fluorescence; DNA; Oligonucleotides; Nucleotides; DNA, Complementary; DNA Probes
PubMed: 36615321
DOI: 10.3390/molecules28010127 -
Current Gene Therapy 2022Gene therapy has made significant development since the commencement of the first clinical trials a few decades ago and has remained a dynamic area of research... (Review)
Review
Gene therapy has made significant development since the commencement of the first clinical trials a few decades ago and has remained a dynamic area of research regardless of obstacles such as immune response and insertional mutagenesis. Progression in various technologies like next-generation sequencing (NGS) and nanotechnology has established the importance of non-- coding segments of a genome, thereby taking gene therapy to the next level. In this review, we have summarized the importance of non-coding elements, highlighting the advantages of using full- length genomic DNA loci (gDNA) compared to complementary DNA (cDNA) or minigene, currently used in gene therapy. The focus of this review is to provide an overview of the advances and the future of potential use of gDNA loci in gene therapy, expanding the therapeutic repertoire in molecular medicine.
Topics: DNA; DNA, Complementary; Genetic Therapy; Genome; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 33874871
DOI: 10.2174/1566523221666210419090357 -
Biophysical Journal Sep 2020Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology,...
Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.
Topics: DNA; DNA Probes; DNA, Single-Stranded; Kinetics; Nucleic Acid Hybridization
PubMed: 32738217
DOI: 10.1016/j.bpj.2020.07.016 -
Biophysical Journal Apr 2023The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent...
The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization and therefore require effective suppression of hybridization errors. A simple and effective method to achieve that is to add blocker strands, also called clamps, to the PCR mixture. These strands bind to the unwanted target sequence, thereby blocking the primer mishybridization. Because of its simplicity, this method is applicable to a broad nucleic-acid-based biotechnology. However, the precise mechanism by which blocker strands suppress PCR errors remains to be understood, limiting the applicability of this technique. Here, we combine experiments and theoretical modeling to reveal this mechanism. We find that the blocker strands both energetically destabilize the mishybridized complex and sculpt a kinetic barrier to suppress mishybridization. This combination of energetic and kinetic biasing extends the viable range of annealing temperatures, which reduces design constraint of the primer sequence and extends the applicability of PCR.
Topics: DNA Primers; Polymerase Chain Reaction; Nucleic Acid Hybridization; Nucleic Acids; Temperature
PubMed: 36823986
DOI: 10.1016/j.bpj.2023.02.028 -
Viruses Jan 2021High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV...
High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 10 copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 10 plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.
Topics: Alphapapillomavirus; Cell Line, Tumor; DNA Probes, HPV; DNA, Viral; Genotype; Human Papillomavirus DNA Tests; Humans; Nucleic Acid Hybridization; Polymerase Chain Reaction; Software
PubMed: 33513748
DOI: 10.3390/v13020188 -
Scientific Reports Jan 2024mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP)...
mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.
Topics: RNA, Messenger; RNA; Biological Assay; DNA Primers; DNA, Complementary
PubMed: 38200031
DOI: 10.1038/s41598-023-49651-8 -
Transboundary and Emerging Diseases Mar 2021To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive...
To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive values of the test. Here, we report the occurrence of contaminations of commercial primers/probe sets with the SARS-CoV-2 target sequence of the RT-qPCR as an example for pitfalls during PCR diagnostics affecting diagnostic specificity. In several purchased in-house primers/probe sets, quantification cycle values as low as 17 were measured for negative control samples. However, there were also primers/probe sets that displayed very low-level contaminations, which were detected only during thorough internal validation. Hence, it appears imperative to pre-test each batch of reagents extensively before use in routine diagnosis, to avoid false-positive results and low positive predictive value in low-prevalence situations. As such, contaminations may have happened more widely, and COVID-19 diagnostic results should be re-assessed retrospectively to validate the epidemiological basis for control measures.
Topics: Benchmarking; COVID-19; COVID-19 Testing; DNA Primers; Equipment Contamination; Germany; Humans; Pandemics; Real-Time Polymerase Chain Reaction; SARS-CoV-2; Sensitivity and Specificity
PubMed: 32536002
DOI: 10.1111/tbed.13684 -
Biotechnology and Applied Biochemistry Aug 2022An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID-19). Here,...
An oligonucleotide DNA probe has been developed for the application in the DNA electrochemical biosensor for the early diagnosis of coronavirus disease (COVID-19). Here, the virus microRNA from the N-gene of severe acute respiratory syndrome-2 (SARS-CoV-2) was used for the first time as a specific target for detecting the virus and became a framework for developing the complementary DNA probe. The sequence analysis of the virus microRNA was carried out using bioinformatics tools including basic local alignment search tools, multiple sequence alignment from CLUSTLW, microRNA database (miRbase), microRNA target database, and gene analysis. Cross-validation of distinct strains of coronavirus and human microRNA sequences was completed to validate the percentage of identical and consent regions. The percent identity parameter from the bioinformatics tools revealed the virus microRNAs' sequence has a 100% match with the genome of SARS-CoV-2 compared with other coronavirus strains, hence improving the selectivity of the complementary DNA probe. The 30 mer with 53.0% GC content of complementary DNA probe 5' GCC TGA GTT GAG TCA GCA CTG CTC ATG GAT 3' was designed and could be used as a bioreceptor for the biosensor development in the clinical and environmental diagnosis of COVID-19.
Topics: Biosensing Techniques; COVID-19; DNA Probes; DNA, Complementary; Genome, Viral; Humans; MicroRNAs; SARS-CoV-2
PubMed: 34378814
DOI: 10.1002/bab.2239 -
Accounts of Chemical Research Dec 2022DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation,...
DNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory's work on developing chemical tools to study DNA repair processes , as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of "two-headed" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a "universal" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.
Topics: Animals; Humans; DNA; DNA Damage; DNA Probes; DNA Repair; Nucleotides
PubMed: 36355579
DOI: 10.1021/acs.accounts.2c00608