DNA (Deoxyribonucleic acid) is the molecule that contains within it all the instructions and information about an organism. This is to say that DNA contains information regarding how the organism will develop, how it lives and reproduces etc. Therefore, the DNA may be described as the blueprint of a living organism.
Given that DNA molecules are found inside the cells, they are too small to be seen with the naked eye. For this reason, a microscope is needed. While it is possible to see the nucleus (containing DNA) using a light microscope, DNA strands/threads can only be viewed using microscopes that allow for higher resolution.
To view the DNA as well as a variety of other protein molecules, an electron microscope is used. Whereas the typical light microscope is only limited to a resolution of about 0.25um, the electron microscope is capable of resolutions of about 0.2 nanometers, which makes it possible to view smaller molecules. This is achieved because electron microscopes use electron beams rather than the visible light used for light microscopes.
For this procedure, the steps involved:
Once the preparation is ready, it is ready for staining
Using the heavy salts allow for higher contrast that makes it possible to view single molecules of the DNA
STEM microscopy has been shown to operate in a dark-field mode thus providing high contrast of biological molecules. Because of its dark-field images, this technique has also been shown to have a great advantage in that it allow for direct visualization of unstained strands of DNA.
Through the high contrast provided, the technique also makes it possible for researchers to be able to identify any problems with the sample. The procedure for this technique is a lot similar to typical electron microscopy for DNA. However, through STEM, researchers obtain both mass and structural information of single-stranded DNA.
Cryo-electron microscopy is one of the techniques that have been shown to be particularly successful in revealing the structure of DNA.
Unlike the transmission electron microscope, Cryo-EM uses frozen samples and electron beams that are gentler to view the sample. This allows researchers to view biological molecules without causing any damage to them in the process.
For this technique, a small amount of the sample in solution is first applied to an EM grid similar to the process used to view DNA strand under electron microscope (EM). The grid with the thin layer is then immersed in liquid ethane (at -180 degrees c) to trap the molecules in water crystal/ice. This ensures that the sample remains is not destroyed when being viewed under the microscope.
Here, it is worth noting that samples prepared for this technique (Cryo-EM samples) tend to be highly sensitive to electron damage. For this reason, low electron doses of about 10–20 e−/Å2 are used to ensure that the sample is not damaged.
While the sample is exposed to low doses of the electrons, the layer of ice around the sample also helps protect the sample during the process.
Through recent advancements in this technique, researchers were able to develop an improved technique of Cryo-EM known as Cryo-electron tomography (CET). Using this technique, it has become possible for researchers to develop 3D structures of various proteins and DNA strands.
Essentially, the process involves the capture of many images of the sample from various angles and using the images to build a 3D structure. Using this technique, researchers have managed to develop and present a variety of 3D images of DNA strands showing the structure of DNA from different angles.
Apart from the electron microscope techniques used to study DNA, methods such as Atomic Force Microscopy (AFM) are also being used for the same purpose. Using this technique, it has become possible for researchers to measure the length of these strands.
Requirements (for AFM)
For this technique, some of the materials required include:
For Double stranded DNA, the same procedure is used but formaldehyde and ammonium acetate is not used.
During imaging, the minimum force was used. This ensured that the cantilever did not lift the sample.
To measure the length of DNA strands using this technique, the images are first enlarged and a fine chain laid along the DNA contours. However, a better way of making the measurements has been shown to involve direct measuring of the top-view of the DNA images in the nanoscope. This method simply involves summing up given points in the image.
The STM can also be used to view DNA molecules. It is capable of imaging objects at atomic levels, which makes it a good tool for viewing DNA molecules.
For this technique, several methods may be used to prepare the sample for imaging, these include:
Method #2 - This method is similar to the first method, but involves vacuum drying the solution and containing it in 10mM ammonium acetate. Sonication was also skipped.
Following imaging, it is possible to identify the DNA through their heights and width. Here, DNA strands can be clearly seen arranged parallel to one another.
One of the most recent methods slightly deviates from the others and involves dissolving DNA in an aqueous solution and placing it in a layer of highly oriented graphite before allowing it to air dry. Here, tungsten-wire tips are used while scanning is carried out under atmospheric conditions. Scanning produces high resolution images of the double helix DNA.
Wolfgang Schonert, original author at GSI.de
As mentioned, DNA (deoxyribonucleic acid) carries within it genetic, hereditary material and resides in the cell nucleus of every organism. Essentially, the strands of DNA are composed of repeated patterns of six molecules which include; deoxyribose (a five carbon sugar) a phosphate group as well as four nitrogenous bases (cytosine (C), thymine (T), adenine (A) and guanine (G)).
The linear order of the bases represents one of the most important features of DNA given that their paring allows for important encoding of information required for development and life of the organism. The structure of DNA (double-helical) is of great significance given that it serves to protect the base atoms.
* Nucleotides are the basic units of DNA. Each nucleotide is composed of a single molecule of sugar, a single phosphate molecule and one nitrogenous base.
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Walther Stoeckenius. Electron Microscopy of DNA Molecules "Stained" With Heavy Metal Salts .
Hele G.Hansma Rober L.Sinsheimer Min-Qia Li and Pau K.Hansma (1992) Atomic force microscopy of single and double-stranded DNA.
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