The Experts below are selected from a list of 316176 Experts worldwide ranked by ideXlab platform
Jaffar Ali Baquir Mohammed - One of the best experts on this subject based on the ideXlab platform.
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A direct detection of Escherichia coli Genomic DNA using gold nanoprobes
Journal of Nanobiotechnology, 2012Co-Authors: Padmavathy Bakthavathsalam, Vinoth Kumar Rajendran, Jaffar Ali Baquir MohammedAbstract:Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli . The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its Genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified Genomic DNA. The method requires less than 30 minutes to complete after Genomic DNA extraction. However, by using unamplified enzymatic digested Genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non- Escherichia coli strains. Conclusion This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.
Ryan B Hayman - One of the best experts on this subject based on the ideXlab platform.
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direct detection of bacterial Genomic DNA at sub femtomolar concentrations using single molecule arrays
Analytical Chemistry, 2013Co-Authors: Linan Song, Dandan Shan, Mingwei Zhao, Brian A Pink, Kaitlin A Minnehan, Lyndsey York, Melissa Gardel, Sean Sullivan, Adrienne A Phillips, Ryan B HaymanAbstract:We report a method for the sensitive measurement of Genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of Genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect Genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL s...
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direct detection of bacterial Genomic DNA at sub femtomolar concentrations using single molecule arrays
Analytical Chemistry, 2013Co-Authors: Linan Song, Dandan Shan, Mingwei Zhao, Brian A Pink, Kaitlin A Minnehan, Lyndsey York, Melissa Gardel, Sean Sullivan, Adrienne A Phillips, Ryan B HaymanAbstract:We report a method for the sensitive measurement of Genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of Genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect Genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).
Padmavathy Bakthavathsalam - One of the best experts on this subject based on the ideXlab platform.
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A direct detection of Escherichia coli Genomic DNA using gold nanoprobes
2013Co-Authors: Padmavathy Bakthavathsalam, Vinoth Kumar Rajendran, Jaffar Ali, Baquir MohammedAbstract:Background: In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results: We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNPoligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its Genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified Genomic DNA. The method requires less than 30 minutes t
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A direct detection of Escherichia coli Genomic DNA using gold nanoprobes
Journal of Nanobiotechnology, 2012Co-Authors: Padmavathy Bakthavathsalam, Vinoth Kumar Rajendran, Jaffar Ali Baquir MohammedAbstract:Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli . The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its Genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified Genomic DNA. The method requires less than 30 minutes to complete after Genomic DNA extraction. However, by using unamplified enzymatic digested Genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non- Escherichia coli strains. Conclusion This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.
Hailin Wang - One of the best experts on this subject based on the ideXlab platform.
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Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides
2016Co-Authors: Junfa Yin, Ning Zhang, Hailin WangAbstract:Structure-based DNA modification analysis provides accurate and important information on Genomic DNA changes from epigenetic modifications to various DNA lesions. However, Genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNAse I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, Genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, Genomic DNA of 5–30 Kb (∼1.6–10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in Genomic DNA
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fluorescence polarization combined capillary electrophoresis immunoassay for the sensitive detection of Genomic DNA methylation
Analytical Chemistry, 2009Co-Authors: Xiaoli Wang, Yuling Song, Maoyong Song, Zhixin Wang, Tao Li, Hailin WangAbstract:Genomic DNA hypomethylation is epigenetically associated with aberrant gene expression and chromosome instability. Here we describe a method for rapid and sensitive detection of Genomic DNA methylation without the need for bisulfite conversion, enzymatic digestion, or PCR amplification. The methylated DNA is first specifically recognized by an anti-5-methylcytosine IgG1 antibody and noncovalently labeled by a monovalent, fluorescently labeled, Fc-specific anti-IgG1 Fab fragment (secondary antibody). The formed immuno-complex of methylated DNA can be efficiently focused and separated from the DNA unbound secondary antibody by capillary electrophoresis (CE). The free fluorescent dye comigrates with the immuno-complex. However, by taking advantage of online laser-induced fluorescence polarization detection (LIFP), the target immuno-complex can be distinguished and accurately measured from the overlapped dye without further separation. The developed method is highly sensitive with a LOD of 0.3 nM and is highl...
Akira F. Peters - One of the best experts on this subject based on the ideXlab platform.
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Extraction of high-quality Genomic DNA from Ectocarpus.
Cold Spring Harbor protocols, 2012Co-Authors: Susana M. Coelho, Delphine Scornet, Sylvie Rousvoal, Nick T. Peters, Laurence Dartevelle, Akira F. PetersAbstract:For some applications, such as genome sequencing and high-throughput genotyping with multiple markers, it is necessary to use high-quality Genomic DNA. This article describes how to obtain several micrograms of high-quality, cesium chloride-purified DNA from 1 g of Ectocarpus filaments. We also recommend using DNA of this quality for quantitative RT-PCR control reactions. However, simpler, more rapid, kit-based methods are preferable for experiments that involve the treatment of large numbers of individuals, such as genotyping large populations with a small number of markers or PCR screening of large populations.
