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Biotechnology Journal Jun 2016Ag(I) is known to interact with cytosine (C) via the formation C-Ag(I)-C complexes. The authors have utilized this concept to design six electrochemical Ag(I) sensors...
Ag(I) is known to interact with cytosine (C) via the formation C-Ag(I)-C complexes. The authors have utilized this concept to design six electrochemical Ag(I) sensors using C-rich DNA probes. Alternating current voltammetry and cyclic voltammetry were used to analyze the sensors. The results show that the dual-probe sensors that require the use of both 5'- and 3'-thiolated DNA probes are not suitable for this application, the differences in probe orientation impedes formation of C-Ag(I)-C complexes. Sensors fabricated with DNA probes containing both thymine (T) and C, independent of the location of the alkanethiol linker, do not response to Ag(I) either; T-T mismatches destabilize the duplex even in the presence of Ag(I). However, sensors fabricated with DNA probes containing both adenine (A) and C are ideal for this application, owing to the formation of C-Ag(I)-C complexes, as well as other lesser known interactions between A and Ag(I). Both sensors are sensitive, specific and selective enough to be used in 50% human saliva. They can also be used to detect silver sulfadiazine, a commonly prescribed antimicrobial drug. With further optimization, this sensing strategy may offer a promising approach for detection of Ag(I) in environmental and clinical samples.
Topics: Biosensing Techniques; Cytosine; DNA Probes; Electrochemical Techniques; Humans; Limit of Detection; Silver; Silver Sulfadiazine
PubMed: 26901685
DOI: 10.1002/biot.201500428 -
Langmuir : the ACS Journal of Surfaces... Mar 2008In this letter, we report the preparation of a compact, functional quantum dot (QD)-DNA conjugate, where the capturing target DNA is directly and covalently coupled to...
In this letter, we report the preparation of a compact, functional quantum dot (QD)-DNA conjugate, where the capturing target DNA is directly and covalently coupled to the QD surface. This enables control of the separation distance between the QD donor and dye acceptor to within the range of the Förster radius. Moreover, a tri(ethylene glycol) linker is introduced to the QD surface coating to effectively eliminate the strong, nonspecific adsorption of DNA on the QD surface. As a result, this QD-DNA conjugate hybridizes specifically to its complementary DNA with a hybridization rate constant comparable to that of free DNAs in solution. We show this system is capable of specific detection of nanomolar unlabeled complimentary DNA at low DNA probe/QD copy numbers via a "signal-on" fluorescence resonance energy transfer (FRET) response.
Topics: Base Sequence; DNA; DNA Adducts; DNA Probes; Molecular Sequence Data; Nucleic Acid Hybridization; Quantum Dots; Spectrometry, Fluorescence
PubMed: 18193909
DOI: 10.1021/la703583u -
Nucleic Acids Research 2007Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually...
Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.
Topics: Base Pair Mismatch; Base Sequence; DNA Probes; Fluorescent Dyes; Genetic Variation; HIV-1; Reverse Transcriptase Polymerase Chain Reaction; Thermodynamics
PubMed: 17693434
DOI: 10.1093/nar/gkm551 -
Molecules (Basel, Switzerland) Feb 2019Simple and rapid detection of DNA single base mismatch or point mutation is of great significance for the diagnosis, treatment, and detection of single nucleotide...
Simple and rapid detection of DNA single base mismatch or point mutation is of great significance for the diagnosis, treatment, and detection of single nucleotide polymorphism (SNP) in genetic diseases. Homogeneous mutation assays with fast hybridization kinetics and amplified discrimination signals facilitate the automatic detection. Herein we report a quick and cost-effective assay for SNP analysis with a fluorescent single-labeled DNA probe. This convenient strategy is based on the efficient quenching effect and the preferential binding of graphene oxide (GO) to ssDNA over dsDNA. Further, a cationic comb-type copolymer (CCC), poly(l-lysine)--dextran (PLL--Dex), significantly accelerates DNA hybridization and strand-exchange reaction, amplifying the effective distinction of the kinetic barrier between a perfect matched DNA and a mismatched DNA. Moreover, in vitro experiments indicate that RAW 264.7 cells cultured on PLL--Dex exhibits excellent survival and proliferation ability, which makes this mismatch detection strategy highly sensitive and practical.
Topics: Animals; Base Pair Mismatch; Biosensing Techniques; Cations; DNA; DNA Probes; Dextrans; Fluorescent Dyes; Mice; Polylysine; RAW 264.7 Cells; Spectrometry, Fluorescence
PubMed: 30764576
DOI: 10.3390/molecules24030575 -
BioTechniques Apr 2018Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming,...
Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Using a targeted synthetic oligonucleotide, such as a gBlocks Gene Fragment, overcomes these drawbacks and provides researchers an accurate and quick solution to standard development. Here, we demonstrate that using a gBlocks fragment as a standard provides comparable sensitivity, reliability, and assay performance to a purified amplicon standard.
Topics: DNA; DNA Primers; DNA Probes; Oligonucleotides; Real-Time Polymerase Chain Reaction; Reproducibility of Results
PubMed: 29661012
DOI: 10.2144/btn-2018-2000 -
Talanta Oct 2021Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked...
Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.
Topics: Biosensing Techniques; COVID-19; Colorimetry; DNA; DNA, Catalytic; DNA, Complementary; Humans; Limit of Detection; Luminescent Measurements; SARS-CoV-2
PubMed: 34215120
DOI: 10.1016/j.talanta.2021.122505 -
Microbial Biotechnology Sep 2011For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic... (Review)
Review
For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α-subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers.
Topics: Archaea; Bacterial Typing Techniques; DNA Primers; DNA Probes; Environmental Microbiology; Methane; Molecular Probe Techniques; Molecular Sequence Data; Phylogeny
PubMed: 21375721
DOI: 10.1111/j.1751-7915.2010.00239.x -
Cellular Oncology : the Official... 2008Multiplex Ligation dependent Probe Amplification (MLPA) is a rapid, simple, reliable and customized method for detection of copy number changes of individual genes at a...
BACKGROUND
Multiplex Ligation dependent Probe Amplification (MLPA) is a rapid, simple, reliable and customized method for detection of copy number changes of individual genes at a high resolution and allows for high throughput analysis. This technique is typically applied for studying specific genes in large sample series. The large amount of data, dissimilarities in PCR efficiency among the different probe amplification products, and sample-to-sample variation pose a challenge to data analysis and interpretation. We therefore set out to develop an MLPA data analysis strategy and tool that is simple to use, while still taking into account the above-mentioned sources of variation.
MATERIALS AND METHODS
MLPAnalyzer was developed in Visual Basic for Applications, and can accept a large number of file formats directly from capillary sequence systems. Sizes of all MLPA probe signals are determined and filtered, quality control steps are performed, and variation in peak intensity related to size is corrected for. DNA copy number ratios of test samples are computed, displayed in a table view and a set of comprehensive figures is generated. To validate this approach, MLPA reactions were performed using a dedicated MLPA mix on 6 different colorectal cancer cell lines. The generated data were normalized using our program and results were compared to previously performed array-CGH results using both statistical methods and visual examination.
RESULTS AND DISCUSSION
Visual examination of bar graphs and direct ratios for both techniques showed very similar results, while the average Pearson moment correlation over all MLPA probes was found to be 0.42. Our results thus show that automated MLPA data processing following our suggested strategy may be of significant use, especially when handling large MLPA data sets, when samples are of different quality, or interpretation of MLPA electropherograms is too complex. It remains, however, important to recognize that automated MLPA data processing may only be successful when a dedicated experimental setup is also considered.
Topics: Algorithms; Colorectal Neoplasms; DNA Probes; DNA, Neoplasm; Gene Dosage; HCT116 Cells; HT29 Cells; Humans; In Situ Hybridization, Fluorescence; Ligase Chain Reaction; Molecular Probe Techniques; Polymerase Chain Reaction; Software; Statistics as Topic
PubMed: 18607067
DOI: 10.3233/clo-2008-0428 -
Research in Microbiology Feb 1992A Listeria monocytogenes-specific, acridinium-ester-labelled DNA probe was evaluated in a chemiluminescent homogeneous protection assay (HPA) for the rapid confirmation...
A Listeria monocytogenes-specific, acridinium-ester-labelled DNA probe was evaluated in a chemiluminescent homogeneous protection assay (HPA) for the rapid confirmation of suspect L. monocytogenes colonies from blood agar plates. The HPA uses an acridinium-ester-labelled chemiluminescent DNA probe in a free-solution hybridization format. After the DNA probe hybridized with the target ribosomal RNA, the acridinium label on the unhybridized probe was inactivated by a chemical differential hydrolysis step. Formation of a hybrid between probe and target was detected in a luminometer after the addition of a detection reagent. The assay can be completed in 30 to 45 min and allows for simultaneous processing of several (50-100) samples. The probe showed 100% sensitivity and 100% specificity for L. monocytogenes when evaluated in the HPA against L. monocytogenes, other Listeria species and other Gram-positive bacteria. The lower detection limit of the HPA was between 10(4) and 10(5) cells. In an evaluation with 296 bacterial colonies isolated from food, the HPA colony confirmation showed 100% agreement with conventional biochemical characterization. HPA will be useful for the rapid confirmation of L. monocytogenes isolated from food and clinical specimens.
Topics: Acridines; Animals; Cattle; DNA Probes; Fishes; Food Microbiology; In Vitro Techniques; Listeria monocytogenes; Listeriosis; Luminescent Measurements; Meat; Vegetables
PubMed: 1410793
DOI: 10.1016/0923-2508(92)90007-b -
Journal of Clinical Microbiology Oct 1993In order to develop a DNA-based assay to identify all Mycobacterium kansasii clinical isolates, a specific DNA probe was isolated in plasmid p6123. A total of 145 M....
In order to develop a DNA-based assay to identify all Mycobacterium kansasii clinical isolates, a specific DNA probe was isolated in plasmid p6123. A total of 145 M. kansasii clinical isolates were collected from several countries and were examined with three probes by DNA hybridization. Of the 145 isolates, 115 (79%) were positive with the previously described probe pMK1-9 (Z. H. Huang, B. C. Ross, and B. Dwyer, J. Clin. Microbiol. 29:2125-2129, 1991), 129 (88%) were positive with the commercial Accu-probe assay (Gen-Probe), and 145 (100%) were positive with the p6123 probe. Southern blot analysis of EcoRI-digested M. kansasii chromosomal DNA with p6123 revealed that all Accu-probe-positive M. kansasii strains exhibited a 3-kb fragment, whereas all Accu-probe-negative M. kansasii strains displayed DNA fragments of variable molecular sizes. These results indicate that, unlike the previously described probes for M. kansasii, the fragment cloned into p6123 identified all 145 biochemically typical strains tested and provides an ideal target for future DNA-based speciation assays.
Topics: Base Sequence; Blotting, Southern; Cloning, Molecular; DNA Probes; Molecular Sequence Data; Nontuberculous Mycobacteria; Plasmids
PubMed: 8253979
DOI: 10.1128/jcm.31.10.2769-2772.1993