scholarly journals Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

2020 ◽  
Author(s):  
Zhen Wang ◽  
Jérôme Maluenda ◽  
Laurène Giraut ◽  
Thibault Vieille ◽  
Andréas Lefevre ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fragile X ◽  
Methylation Status ◽  
Single Molecules ◽  
High Sensitivity ◽  
Base Modification ◽  
Dna And Rna ◽  
Magnetic Tweezer ◽  
Base Modifications ◽  
Sensitivity Specificity

AbstractAccurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5‘UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

scholarly journals Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Zhen Wang ◽  
Jérôme Maluenda ◽  
Laurène Giraut ◽  
Thibault Vieille ◽  
Andréas Lefevre ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fragile X ◽  
Methylation Status ◽  
Single Molecules ◽  
High Sensitivity ◽  
E Coli ◽  
Dna And Rna ◽  
Magnetic Tweezer ◽  
Base Modifications ◽  
Sensitivity Specificity

AbstractAccurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5’UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.

scholarly journals Single-molecule optical absorption imaging by nanomechanical photothermal sensing

2018 ◽  
Vol 115 (44) ◽  
pp. 11150-11155 ◽  
Author(s):  
Miao-Hsuan Chien ◽  
Mario Brameshuber ◽  
Benedikt K. Rossboth ◽  
Gerhard J. Schütz ◽  
Silvan Schmid
Keyword(s):  
Single Molecule ◽  
Shot Noise ◽  
Signal To Noise Ratio ◽  
Local Heating ◽  
Single Molecules ◽  
High Sensitivity ◽  
Temperature Sensitive ◽  
Signal To Noise ◽  
Promising Alternative ◽  
Noise Ratio

Absorption microscopy is a promising alternative to fluorescence microscopy for single-molecule imaging. So far, molecular absorption has been probed optically via the attenuation of a probing laser or via photothermal effects. The sensitivity of optical probing is not only restricted by background scattering but it is fundamentally limited by laser shot noise, which minimizes the achievable single-molecule signal-to-noise ratio. Here, we present nanomechanical photothermal microscopy, which overcomes the scattering and shot-noise limit by detecting the photothermal heating of the sample directly with a temperature-sensitive substrate. We use nanomechanical silicon nitride drums, whose resonant frequency detunes with local heating. Individual Au nanoparticles with diameters from 10 to 200 nm and single molecules (Atto 633) are scanned with a heating laser with a peak irradiance of 354 ± 45 µW/µm2 using 50× long-working-distance objective. With a stress-optimized drum we reach a sensitivity of 16 fW/Hz1/2 at room temperature, resulting in a single-molecule signal-to-noise ratio of >70. The high sensitivity combined with the inherent wavelength independence of the nanomechanical sensor presents a competitive alternative to established tools for the analysis and localization of nonfluorescent single molecules and nanoparticles.

scholarly journals The Single Molecule Imaging Approach to Membrane Protein Stoichiometry

2012 ◽  
Vol 18 (4) ◽  
pp. 771-780 ◽  
Author(s):  
Richard Hallworth ◽  
Michael G. Nichols
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Molecules ◽  
High Sensitivity ◽  
Direct Observations ◽  
Subunit Stoichiometry ◽  
Technical Advances ◽  
Methodological Considerations ◽  
Visualization Techniques ◽  
Fluorescent Molecules

AbstractRecent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

scholarly journals Recent advances in the detection of base modifications using the Nanopore sequencer

2019 ◽  
Vol 65 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Liu Xu ◽  
Masahide Seki
Keyword(s):  
Single Molecule ◽  
Statistical Tests ◽  
Regulation Of Gene Expression ◽  
Detection Methods ◽  
Open Chromatin ◽  
Rna Molecules ◽  
Base Modification ◽  
Dna And Rna ◽  
Long Read ◽  
Nanopore Sequencer

Abstract DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

scholarly journals Quantification of purified endogenous miRNAs with high sensitivity and specificity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Soochul Shin ◽  
Yoonseok Jung ◽  
Heesoo Uhm ◽  
Minseok Song ◽  
Soomin Son ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression Profiles ◽  
Thermus Thermophilus ◽  
High Sensitivity ◽  
Transcriptional Level ◽  
Protein Coding ◽  
Copy Numbers ◽  
Endogenous Mirnas ◽  
Average Copy ◽  
Sensitivity Specificity

AbstractMicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3′-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.

TO SEPARATELY ASSESS THE DIAGNOSTIC POTENTIAL OF TVS & MRI IN THE TERMS OF SENSITIVITY AND SPECIFICITY BY CORRELATING THE IMAGING FINDINGS WITH HISTOPATHOLOGY

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Suraj Mathur
Keyword(s):  
Transvaginal Ultrasound ◽  
High Sensitivity ◽  
Imaging Evaluation ◽  
Medical College ◽  
Conventional Mri ◽  
Diagnostic Potential ◽  
Adc Mapping ◽  
Malignant Lesions ◽  
Sensitivity Specificity

This prospective study was done in the Department of Radio diagnosis Govt. Medical College, Kozhikode. A total of 65 patients who were referred to our department with clinical suspicion of endometrial lesions and incidentally detected endometrial lesions on ultrasonography underwent transvaginal ultrasound and subsequent Imaging evaluation of pelvis MRI has very high sensitivity (95%) and specificity (98%) and is almost as accurate (97%) as histopathology in differentiating benign from malignant lesions. Addition of DWI with ADC mapping to conventional MRI increases its accuracy even more. However there is inherent limitation to MRI in detecting carcinoma in situ and micrometastasis. Keywords: TVS, MRI, Sensitivity, Specificity, Histopathology.

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

scholarly journals Use of β-D-glucan in diagnosis of suspected Pneumocystis jirovecii pneumonia in adults with HIV infection

2021 ◽  
pp. 095646242110222
Author(s):  
Thomas Juniper ◽  
Chris P Eades ◽  
Eliza Gil ◽  
Harriet Fodder ◽  
Killian Quinn ◽  
...  
Keyword(s):  
Predictive Value ◽  
Diagnostic Tests ◽  
Elevated Serum ◽  
Clinical Information ◽  
High Sensitivity ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Alternative Diagnosis ◽  
Positive Threshold ◽  
Sensitivity Specificity

Objectives: An elevated serum (1-3)-β-D-glucan (BDG) concentration has high sensitivity for a diagnosis of Pneumocystis pneumonia (PCP) in people with HIV (PWH). At the current manufacturer-recommended positive threshold of 80 pg/mL (Fungitell), specificity for PCP is variable and other diagnostic tests are required. We evaluated the utility of serum BDG for diagnosis of suspected PCP in PWH at three inner-London hospitals to determine BDG concentrations for diagnosis and exclusion of PCP. Methods: From clinical case records, we abstracted demographic and clinical information and categorised patients as having confirmed or probable PCP, or an alternative diagnosis. We calculated sensitivity, specificity and positive predictive value (PPV) of serum BDG concentrations >400 pg/mL and negative predictive value (NPV) of BDG <80 pg/mL. Results: 76 patients were included; 29 had laboratory-confirmed PCP, 17 had probable PCP and 30 had an alternative diagnosis. Serum BDG >400 pg/mL had a sensitivity of 83%, specificity of 97% and PPV 97% for diagnosis of PCP; BDG <80 pg/mL had 100% NPV for exclusion of PCP. Conclusions: In PWH with suspected PCP, BDG <80 pg/mL excludes a diagnosis of PCP, whereas BDG concentrations >400 pg/mL effectively confirm the diagnosis. Values 80–400 pg/mL should prompt additional diagnostic tests.

scholarly journals The Child Behavior Checklist as a Screening Instrument for PTSD in Refugee Children

Children ◽  
2021 ◽  
Vol 8 (6) ◽  
pp. 521
Author(s):  
Ina Nehring ◽  
Heribert Sattel ◽  
Maesa Al-Hallak ◽  
Martin Sack ◽  
Peter Henningsen ◽  
...  
Keyword(s):  
Child Behavior ◽  
Child Behavior Checklist ◽  
High Sensitivity ◽  
Roc Curves ◽  
Structured Interview ◽  
Screening Instrument ◽  
Screening Tools ◽  
Refugee Children ◽  
Post Traumatic Stress ◽  
Sensitivity Specificity

Thousands of refugees who have entered Europe experienced threatening conditions, potentially leading to post traumatic stress disorder (PTSD), which has to be detected and treated early to avoid chronic manifestation, especially in children. We aimed to evaluate and test suitable screening tools to detect PTSD in children. Syrian refugee children aged 4–14 years were examined using the PTSD-semi-structured interview, the Kinder-DIPS, and the Child Behavior Checklist (CBCL). The latter was evaluated as a potential screening tool for PTSD using (i) the CBCL-PTSD subscale and (ii) an alternative subscale consisting of a psychometrically guided selection of items with an appropriate correlation to PTSD and a sufficient prevalence (presence in more than 20% of the cases with PTSD). For both tools we calculated sensitivity, specificity, and a receiver operating characteristic (ROC) curve. Depending on the sum score of the items, the 20-item CBCL-PTSD subscale as used in previous studies yielded a maximal sensitivity of 85% and specificity of 76%. The psychometrically guided item selection resulted in a sensitivity of 85% and a specificity of 83%. The areas under the ROC curves were the same for both tools (0.9). Both subscales may be suitable as screening instrument for PTSD in refugee children, as they reveal a high sensitivity and specificity.

scholarly journals Evaluation of HiCrome KPC Agar for the Screening of Carbapenem-Resistant Enterobacterales Colonization in the ICU Setting of a Tertiary Care Hospital

2021 ◽  
Author(s):  
Ashoka Mahapatra ◽  
K Nikitha ◽  
Sutapa Rath ◽  
Bijayini Behera ◽  
Kavita Gupta
Keyword(s):  
Tertiary Care ◽  
High Sensitivity ◽  
Care Hospital ◽  
Predictive Values ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Carbapenem Resistant ◽  
Rectal Swabs ◽  
Control And Prevention ◽  
Indian Setting ◽  
Sensitivity Specificity

Abstract Background Spread of carbapenem-resistant Enterobacterales (CRE) is a significant concern in intensive care unit (ICU) settings. Approaches to routine screening for CRE colonization in all ICU patients vary depending on institutional epidemiology and resources. The present study was aimed to evaluate the performance of HiCrome Klebsiella pneumoniae carbapenemase (KPC) agar for the detection of CRE colonization in ICU settings taking the Centers for Disease Control and Prevention (CDC) recommended method as reference. Methods Two-hundred and eighty rectal swabs (duplicate) from 140 patients were subjected to CRE detection in HiCrome KPC agar and MacConkey agar (CDC criteria). Results Using CDC method, total 41 CRE isolates were recovered comprising of 29 E scherichia coli, 11 Klebsiella, and 1 Enterobacter spp. On the other hand, 49 isolates of CRE recovered from 140 rectal swabs using HiCrome KPC agar, out of which 33 were E. coli, 15 Klebsiella, and 1 Enterobacter sp. Statistical Analysis Sensitivity, specificity, negative, and positive predictive values of CRE screening by HiCrome KPC agar were found to be 100% (91.4–100), 91.9% (84.8–95.8), 83.6% (70.9–91.4), and 100% (95.9–100), respectively, taking the CDC recommended method as reference. Conclusion HiCrome KPC agar has high sensitivity in screening CRE colonization. Further studies are needed to establish its applicability for detecting the predominant circulating carbapenemases in the Indian setting.

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