Browse
Submit Data
Databases
API
Help

Dataset Information

38 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single DNA molecule patterning for high-throughput epigenetic mapping.

ABSTRACT: We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states.

SUBMITTER: Cerf A

PROVIDER: S-EPMC3773698 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Single DNA molecule patterning for high-throughput epigenetic mapping.

Cerf Aline A Cipriany Benjamin R BR Benítez Jaime J JJ Craighead Harold G HG

Analytical chemistry 20111013 21

We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are ...[more]

PMID: 21981444

Similar Datasets

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.

Project description:DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500-1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

| S-EPMC9458417 | biostudies-literature

High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging.

Project description:Single-molecule studies of protein-DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains-a method for organizing arrays of DNA molecules on a fluid lipid bilayer-has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900,000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3-a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.

| S-EPMC4624423 | biostudies-literature

Single molecule high-throughput footprinting of small and large DNA ligands.

Project description:Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.

| S-EPMC5563512 | biostudies-literature

High-throughput single-molecule optofluidic analysis.

Project description:We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

| S-EPMC3075913 | biostudies-literature

High-throughput single-molecule telomere characterization.

Project description:We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines.

| S-EPMC5668947 | biostudies-literature

High-throughput mapping of regulatory DNA.

Project description:Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell-specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair-resolution functional motifs of regulatory elements.

| S-EPMC5108523 | biostudies-literature

High-throughput single-molecule mapping links subtelomeric variants and long-range haplotypes with specific telomeres.

Project description:Accurate maps and DNA sequences for human subtelomere regions, along with detailed knowledge of subtelomere variation and long-range telomere-terminal haplotypes in individuals, are critical for understanding telomere function and its roles in human biology. Here, we use a highly automated whole genome mapping technology in nano-channel arrays to analyze large terminal human chromosome segments extending from chromosome-specific subtelomere sequences through subtelomeric repeat regions to terminal (TTAGGG)n repeat tracts. We establish detailed maps for subtelomere gap regions in the human reference sequence, detect many new large subtelomeric variants and demonstrate the feasibility of long-range haplotyping through segmentally duplicated subtelomere regions. These features make the method a uniquely valuable new tool for improving the quality of genome assemblies in complex DNA regions. Based on single molecule mapping of telomere-terminal DNA fragments, we provide proof of principle for a novel method to estimate telomere lengths linked to distinguishable telomeric haplotypes; this single-telomere genotyping method may ultimately enable delineation of human cis elements involved in telomere length regulation.

| S-EPMC5605236 | biostudies-literature

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion.

Project description:Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.

| S-EPMC3384352 | biostudies-literature

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Project description:Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called 'DNA skybridge'. The DNA skybridge is constructed in a 3D structure with 4 μm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

| S-EPMC6765118 | biostudies-literature

Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule Spectroscopy.

Project description:We experimentally investigated several intramolecular coordinate and environmental changes as potential causes of single-molecule fluorescence spectral heterogeneities (smFSH). We developed a high-throughput single-molecule spectroscopy method to analyze more than 5000 single-molecule emission spectra from each of 9 commonly used fluorophores with different structural rigidities and deposited on substrates with different polarities. We observed an unexpectedly high smFSH from structurally rigid Rhodamine B compared with a structurally flexible Cyanine dye-Alexa Fluor 647. Based on experimentally measured smFSH, we ruled out the system's noise uncertainty, single-molecule spectral diffusion, and environmental polarity as the primary causes of the high smFSH. We found that the rotational flexibility of N,N-dialkylated groups contributed to the smFSH. With the high smFSH observed in structurally more rigid model fluorophores, we speculated that other intramolecular coordinate and environmental changes might also contribute to the high smFSH in Rhodamines.

| S-EPMC8607629 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data