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Genomic DNA in Prokaryotes00:46

Genomic DNA in Prokaryotes

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The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
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Genomic DNA in Eukaryotes00:58

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Eukaryotes have large genomes compared to prokaryotes. To fit their genomes into a cell, eukaryotic DNA is packaged extraordinarily tightly inside the nucleus. To achieve this, DNA is tightly wound around proteins called histones, which are packaged into nucleosomes that are joined by linker DNA and coil into chromatin fibers. Additional fibrous proteins further compact the chromatin, which is recognizable as chromosomes during certain phases of cell division.
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Atomic Fluorescence Spectroscopy01:29

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Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which...
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Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal and vicinal coupling are observed. For example, consider the COSY spectrum of ethyl acetate, where its 1D proton NMR spectrum is plotted along the vertical and horizontal axes with their corresponding chemical shift scale. Three spots on the diagonal corresponding to the three peaks in the 1D proton spectrum are called diagonal peaks. The COSY...
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Genomics is the science of genomes: it is the study of all the genetic material of an organism. In humans, the genome consists of information carried in 23 pairs of chromosomes in the nucleus, as well as mitochondrial DNA. In genomics, both coding and non-coding DNA is sequenced and analyzed. Genomics allows a better understanding of all living things, their evolution, and their diversity. It has a myriad of uses: for example, to build phylogenetic trees, to improve productivity and...
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Correlation means that there is a relationship between two or more variables (such as ice cream consumption and crime), but this relationship does not necessarily imply cause and effect. When two variables are correlated, it simply means that as one variable changes, so does the other. We can measure correlation by calculating a statistic known as a correlation coefficient. A correlation coefficient is a number from -1 to +1 that indicates the strength and direction of the relationship between...
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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
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Fluorescence Correlation Spectroscopy on Genomic DNA in Living Cells.

Cameron Hodges1, Jens-Christian Meiners2

  • 1Department of Biophysics, University of Michigan, Ann Arbor, MI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 30, 2018
PubMed
Summary

This chapter details quantitative Fluorescence Correlation Spectroscopy (FCS) protocols for measuring DNA dynamics in living cells. It addresses challenges like photobleaching for accurate biomolecular transport analysis.

Keywords:
Chromosomal DNAFluorescence correlation spectroscopyLive cell microscopyPhotobleaching

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Cellular Dynamics

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is vital for studying biomolecular transport.
  • Applying FCS in living cells presents significant experimental challenges and artifacts.
  • Quantitative measurements of DNA dynamics within cells are crucial for understanding cellular processes.

Purpose of the Study:

  • To provide standardized protocols for quantitative FCS measurements of DNA in living eukaryotic and prokaryotic cells.
  • To guide researchers in overcoming experimental difficulties associated with in vivo FCS.
  • To enable accurate characterization of DNA diffusion and transport within the cellular environment.

Main Methods:

  • Detailed protocols for sample preparation and fluorescent dye selection/characterization.
  • Methods for FCS data acquisition within living cells.
  • Development and application of data analysis techniques, including photobleaching compensation.

Main Results:

  • Demonstration of reliable quantitative FCS measurements of DNA in various cell types.
  • Successful compensation for photobleaching artifacts, enhancing data accuracy.
  • Generation of quantitatively meaningful FCS spectra from intracellular DNA.

Conclusions:

  • Established protocols enable robust in vivo FCS analysis of DNA dynamics.
  • Photobleaching compensation is essential for accurate quantitative results.
  • This work facilitates deeper understanding of DNA transport and behavior in living cells.