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While you are well familiar with the term ‘DNA’ the isolation of DNA may seem a bit bewildering at first. The theory and know-how are a crucial part of the Life Science subject. A broad understanding of life processes at the molecular and cellular levels can give you a deep comprehension of the DNA structure. If that’s already holding your attention, studying the thesis from scratch is the correct thing to do. How about sparing a few minutes and getting your basics right? Begin by spending a moment or two on these blogs.
Before we get into the isolation of DNA and its extraction process, here are two foremost factors.
DNA isolation is the first crucial step in swotting a particular DNA chain. An intricate DNA population and genome structure are broken down with the help of this essential technique. In a snippet, the process involves separating a component from the cortex and then purifying the nucleic acid. Interestingly, the crude measure has various sources. As mentioned above, the process of DNA isolation can be done on any organism, including hair, blood, nails, sperm, bloodstains, urine, saliva, bones, nails and more.
The process and the purpose for the isolation of DNA vary. The technique used to isolate DNA will be conditioned on several factors such as the sample size, age and source. However, despite the difference in approach, the process of isolation remains the same. Biotechnology adopts the procedure to diagnose the pressing problems of today revolving around life-threatening diseases like cancer in the medicinal field. Scientists are major DNA hunters to recognize different species and understand how they are controlled.
Speaking of forensic science, experts conduct DNA isolation to identify individuals in case of any unpleasant circumstances of robbery, rape, war or accident. Currently, DNA isolation is a routine procedure in the mentioned spheres. Did you know when the first DNA isolation was conducted? Swiss Biologist and Physician, Johannes Friedrich Miescher, was the first to isolate nucleic acid in 1869.
The DNA isolation steps involve five major steps.
For starters, DNA extraction is completely different from isolation. The latter is a general term for the whole procedure, and DNA extraction is just one part of it. To put it simply, it is the methodology used to isolate and purify the DNA in a sample. In the isolation process, there may be a tad of contamination. In contrast, extraction decontaminates the DNA with some physical or chemical methods. DNA extraction is pivotal in the isolation process. It determines the shape, size and function of the DNA sample. The extraction also stands as the baseline for the research, diagnosis, and decision-making. The DNA extraction protocol majorly involves using a suitable method.
The DNA extraction method can vary depending upon the sample. If we broadly categorize the mechanism, there are two types of extraction methods.
The solution-based formula is further divided into organic and inorganic extraction. The Organic solvent-based DNA extraction method involves toxic elements like phenol and chloroform. The process has an increased risk of contamination and therefore is less recommended. Also, the procedure is highly time-consuming.
The inorganic DNA extraction method is classified as the proteinase K and salting-out procedure. Erstwhile encourages high DNA yield but can be cumbersome. Also, the system is prone to the low stability of the enzyme, and it requires good maintenance in a cold chain. The salting-out DNA extraction method is non-toxic and safe. The process uses salts like potassium acetate, sodium chloride and ammonium acetate, which help in the extraction. Though a satisfactory yield, the purity level falls short.
This method can be automated and has a high throughput. The entire process can be completed in one tube. Therefore, the risk of contamination remains negligible. Solid-phase is mostly beneficial for forensics; however, it comes with the drawback of being expensive. It involves the magnetic and paper procedure. With the magnetic extraction technique, the DNA is divided under the magnetic field and requires a DNA extraction buffer.
The paper extraction manner is commonly used to isolate DNA from plant sources. The method uses filter paper to absorb the DNA from the sample speedily.
DNA extraction involves three major steps.
DNA is released by breaking down of the cell wall and nucleus, and mechanical interference breaks open the cells. The tissue is cut open into small pieces with the help of a small blender or a mortar and pestle. This is hard enough for the tough cell walls. In the case of soft cell walls, it is broken down with enzymes and detergents.
It is a crucial step that separates the DNA from the cellular debris. It involves chemicals like isopropanol, sodium acetate and ethanol. Sodium ions neutralise the negative ions on the DNA sample and work on decreasing the solubility in water. Meanwhile, ethanol and isopropanol are added, which causes the DNA to precipitate.
Once perception sets the DNA from the aqueous stage, it is washed and rinsed with any type of alcohol. All the contamination, unwanted material and cellular debris are separated from the DNA. The refined DNA is then dissolved in distilled water for storage.
When you research DNA isolation and extraction, you will often encounter the term ‘DNA spooling’. To put it simply, it is an exercise that isolates long DNA strands from a solution. DNA is super sticky and hence can easily adhere to any surface. When the DNA strand is spooled, it looks closer to a white strand which is pure DNA. A glass stirring rod or a wooden spooling stick moves the molecules away from ethanol.
For uninterrupted DNA spooling, tilt the mini beaker a little bit but be very cautious about not mixing the layers. Position the wooden spooling stick at the point of an interface of the solution. The mini beaker must be inclined to the right 45°. Then plunge the stick into the DNA layer, spool it and pull out the DNA with the spooling stick. When the strands are spooled tighter, the ethanol will be pushed out. If the mini beaker still has DNA, use another stick to spool more DNA. For a large DNA sample, allow more ethanol to interact with DNA. Once the DNA spooling is done, it must be scraped into a tube immediately.
Conclusively, the purpose for the isolation of DNA and extraction is the same; to derive unadulterated DNA. A pure DNA sample has a ratio of absorbance at 260 nm and 280 nm. When you find the ratio of < 1.8, it is a clear indication that the sample is contaminated. The DNA extraction protocol and method take care of fragmenting the DNA concentration.
Our detailed DNA guide was to help you understand the extensive topic. If you find it gripping and need further assistance, at Atria University, you can get a good hold of the subject and gain some practical knowledge in our Life Science Major Program. Even a day in the life of a DNA specialist can make you a fortune.
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