Dna Fiber Analysis
Dna Fiber Analysis - Related Videos
07:18
Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique
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Cited by 73 •
2011
DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.
08:47
DNA Methylation Analysis
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2023
Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the methylation of clusters of methylation sites, called “CpG islands”, near promoters and other gene regulatory elements may contribute to the stable silencing of genes, for example, during epigenetic processes such as genomic imprinting and X-chromosome inactivation. At the same time, aberrant CpG methylation has been shown to be associated with...
08:28
Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers
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Cited by 6 •
2017
A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.
12:34
DNA Methylation: Bisulphite Modification and Analysis
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Cited by 75 •
2011
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
10:31
Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers
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2017
This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.
FAQs
Sequential thymidine analog labeling creates two temporally distinct fluorescent segments within newly synthesized DNA. CldU and IdU are supplied in separate labeling periods, so the relative lengths and continuity of their tracks indicate how far replication forks progressed during each interval. This temporal pattern allows researchers to compare fork movement directly and identify changes in replication dynamics.
Unequal track lengths on the two sides of a replication region indicate fork asymmetry, whereas interrupted or absent extension can identify stalled forks. These patterns provide single-molecule evidence of disrupted replication progression rather than only a bulk cellular measurement. In genetics research, the findings help evaluate replication stress and mechanisms associated with genome instability.
The distances between labeled replication tracks provide information about the arrangement of replication origins along individual DNA molecules. Comparing these intervals reveals origin spacing and can show whether initiation patterns are altered between experimental conditions. This measurement complements fork-speed and fork-symmetry analyses, allowing researchers to examine both where replication begins and how efficiently it proceeds.
Cells are first exposed sequentially to different thymidine analogs, such as CldU and IdU. DNA is then extracted gently so replication structures remain interpretable and stretched onto microscope slides. Fluorescent antibodies detect the incorporated analogs, after which the resulting tracks are examined microscopically. Track patterns can be measured to assess fork progression, origin spacing, asymmetry, and stalling.
Researchers use this approach when they need direct information about replication behavior in individual DNA molecules, particularly after cellular conditions produce replication stress. The assay can distinguish slowed or stalled forks, altered origin spacing, and asymmetric fork movement. These measurements help connect a cellular response with specific changes in replication dynamics and clarify how replication and repair factors influence genome stability.
In genetics, the method helps characterize defects in factors that regulate DNA replication or repair by revealing their effects on fork movement and stability. Comparisons between experimental and reference cells can expose abnormal stalling, asymmetry, or origin organization. Such results support investigation of genome instability and disease-associated replication defects while providing a mechanistic view of altered DNA duplication.