Video of Corona Virus RNA Extraction by Silica Gel column

[Virtual Presenter] Please reword the text below in complete sentences. Thank you..

DNA Isolation. [image].

[Audio] The learning objectives for today's session are to define DNA isolation or extraction, list different methods of isolating DNA, describe one such method, and explore its various uses. These objectives will serve as our guide throughout this presentation..

[Audio] DNA isolation/extraction is a routine procedure used in molecular biology laboratories. This starting point is crucial in numerous applications ranging from fundamental research to routine diagnostic and therapeutic decision-making. The DNA extraction process frees the DNA from cells and then separates it from cellular fluid and proteins. As a result, what remains is pure DNA. This solution of pure DNA is necessary for genetic analysis, which can be applied in various fields such as scientific and research purposes, medical diagnostic purposes, forensic science, paternity, and more..

[Audio] DNA extraction, DNA isolation, and DNA purification are often used interchangeably, but they serve slightly different purposes. DNA isolation aims to obtain as much DNA as possible from a sample. On the other hand, DNA purification is used to remove contaminants from the extracted DNA. DNA extraction, meanwhile, refers to a specific method that achieves both isolation and purification..

[Audio] The DNA extraction process is a crucial step in many molecular biology experiments. Breaking open the cell membrane and releasing the genetic material is necessary to study or analyze DNA. The extracted DNA can then be used for various applications such as PCR, sequencing, cloning, and more..

[Audio] DNA isolation can occur from a vast array of sources, including living organisms such as humans, animals, plants, and microorganisms. These sources can provide DNA for various applications, including research, diagnostics, and forensic analysis. In addition, DNA can be extracted from deceased individuals, such as those who have passed away recently or even ancient remains. This versatility makes DNA isolation a crucial tool in many fields..

[Audio] DNA is found within the cells. Eukaryotes have their DNA enveloped within the nucleus, whereas prokaryotes have it situated inside the cytoplasm. Prior to commencing the DNA extraction process, it is essential to comprehend that cells contain additional components alongside DNA. These include enzymes, proteins, organic, and inorganic compounds, all of which must be eliminated prior to extracting the DNA..

[Audio] The process of DNA extraction varies on many factors, including the target molecule or sample type. Genomic DNA extraction procedures differ from those used to extract mRNA or plasmids. Furthermore, the extraction of genomic DNA requires different techniques depending on the sample type, such as blood, tissue, or plant..

[Audio] The first step in DNA extraction is lysis, where cells are broken open to release their contents, including DNA. This is typically done using a lysis buffer, which helps to disrupt the cell membranes and release the genetic material..

[Audio] In the initial stage of DNA extraction, we need to break open the cell walls and membranes to release the DNA. This process is called lysis. There are two ways to achieve this - physically or chemically. To break down the cell walls and membranes, we can use either or both physical and chemical methods. However, when it comes to breaking down the nuclear membrane, chemical methods are typically preferred. By releasing the DNA from its protective structures, we're one step closer to isolating it..

[Audio] There are two main methods of cell disruption, both physical and chemical. Physical methods involve mechanically breaking open the cell, including using a blender, mortar and pestle, cutting the specimen into smaller pieces, or employing a sonicator. These methods are particularly useful for plant cells with tough walls. On the other hand, chemical methods utilize detergents or enzymes to break apart biological membranes. Detergents, such as SDS, facilitate rapid breakdown of biological membranes, whereas enzymes, including Proteinase K, Cellulase, Lyticase, and Lysozyme, are more effective at extracting DNA since they directly break down protein bonds..

[Audio] After the cell lysis step, the DNA is mixed with other cell parts, forming a cell extract. This mixture contains various substances like detergents, proteins, and reagents used during the lysis process. Precipitation is then employed to separate the DNA from this mixture. Sodium ions are added to neutralize the negative charge on the DNA molecules, making them less water-soluble and more stable. Next, alcohol such as ethanol or isopropanol is added, causing the DNA to precipitate out of the solution because it is insoluble in alcohol. To obtain a clean sample of DNA, it is essential to remove as much of the cellular debris as possible. This can be achieved through various methods, including the addition of proteases, which degrade any DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample..

[Audio] The precipitation step is crucial because it allows us to obtain DNA in its pure form. Without it, the presence of cellular debris such as proteins, lipids, polysaccharides, and other organic and inorganic compounds can compromise the integrity of the DNA. These contaminants can not only interfere with DNA analysis methods but also reduce the quality of the DNA, ultimately leading to a shorter storage life..

[Audio] After the separation of DNA during the process of precipitation, the DNA is now located in the ethanol area. It is rinsed with alcohol, either with isopropanol or absolute ethanol, in a process known as purification. This step removes all of the remaining cellular debris and unwanted material. As a result, the DNA is now completely purified. It is typically dissolved in water again for convenient storage and handling before further processing..

[Audio] After completing the three steps of DNA extraction, we need to assess the concentration and quality of the DNA. This can be done using a spectrophotometer to measure concentration and purity, or gel electrophoresis to check for DNA presence and quality. Once this assessment is complete, we can proceed with other molecular analyses, including PCR, sequencing, fingerprinting, or cloning..

[Audio] Now that we have learned about the different steps in the extraction methods, it is time to familiarize ourselves with the different methods used for extracting DNA. DNA extraction methods are broadly categorized into two: the chemical and the physical. The three steps for DNA extraction remain the same in all the different types of methods. The physical extraction method makes use of magnetic beads or different kinds of paper. The chemical DNA extraction method, also called the solution-based extraction method, is subdivided into organic and inorganic. The organic solvent-based DNA extraction method is based on the use of organic substances such as phenol and chloroform, but due to the harmful nature of both phenol and chloroform, the method is restricted. Nevertheless, the phenol and chloroform DNA extraction method is one of the best methods among all. This method is also known as the phenol-chloroform-isoamyl-alcohol method. Among the inorganic DNA extraction methods, two are the most popular: proteinase K and the use of salt. The proteinase K DNA extraction method facilitates high DNA yield, but the method is time-consuming, and if not maintained well in a cold chain, the proteinase K cannot be utilized for a longer period of time. The salting-out method is a safer method compared to the phenol-chloroform method. The use of salt, such as sodium chloride, potassium acetate, and ammonium acetate, helps in the DNA extraction. However, the method is more aggressive in combination with proteinase K. The next method is the silica-based method, which works on the unique chemistry of interaction between the silica and the DNA..

[Audio] Now that we've covered the fundamental steps of DNA extraction, let's examine the various extraction methods. These methods are broadly categorized into physical and chemical techniques, but the essential three steps remain the same for all. Chemical Methods, also referred to as solution-based methods, employ solvents like phenol and chloroform, which are highly effective but toxic, limiting their usage. This method is also known as the phenol-chloroform-isoamyl alcohol method and is one of the best for DNA extraction. Inorganic methods utilize safer alternatives to organic solvents, including the Proteinase K method, which yields high-quality DNA but requires cold storage for long-term use. The salting-out method employs salts like sodium chloride, potassium acetate, or ammonium acetate for DNA extraction. This method is safer than the phenol-chloroform method and functions well when combined with Proteinase K. Finally, the silica-based method relies on the unique interaction between silica and DNA, distinguishing it from other extraction techniques..

[Audio] The process of extracting Coronavirus RNA from a sample involves several steps. A swab is used to collect a sample from a patient's nose or throat, which is then placed in a special liquid called a viral transport medium to keep the virus alive. The sample is mixed with a chemical called lysis buffer, which breaks down the cells and releases the genetic material inside. This mixture is then passed through a silica gel column, which separates the genetic material from other substances. Finally, the purified RNA is ready to be used in a PCR test to detect the presence of the Coronavirus..

Bacterial Sample.

[Audio] In this step, we are focusing on another inorganic method, which is a silica-based DNA extraction technique. This protocol is specifically designed for bacterial samples, and we will use it to extract the DNA from our sample..

[Audio] The silica column-based DNA extraction method differs significantly from other techniques, such as the phenol-chloroform and Proteinase K method, which necessitate multiple centrifugation steps and phase separation. This method hinges on the chemical interaction between silica and DNA, wherein positively charged silica binds to negatively charged DNA, retaining it during centrifugation. Initially described by McCormick in 1989, the concept was initially developed by Vogelstein in 1979 for DNA purification. Currently commercially available, this method is extensively employed in diagnostic laboratories..

[Audio] The process starts with cell lysis, where cells are broken open to release their contents, including the genetic material. This step is crucial in releasing the RNA from the cellular structure. Next, DNA binding occurs, where the released RNA binds to the silica gel column. This step is essential in capturing the target RNA molecule. Then, washing the sample is necessary to remove any impurities or contaminants that may interfere with the subsequent steps. Finally, elution of the DNA takes place, where the bound RNA is released from the silica gel column, ready for further analysis such as PCR testing..

[Audio] The process starts by incubating the bacterial cell suspension with a cell lysis buffer and a small amount of Proteinase K. This breaks down the cell membrane and nuclear envelope, allowing the proteins to be digested. Then, the Nucleospin bead tube contains beads that mechanically disrupt the cell wall, releasing the DNA. By following the Nucleospin protocol, we ensure efficient DNA extraction..

[Audio] So, the binding process involves adjusting the DNA binding capacity by adding a buffer to re-suspend the glass beads properly. Then, the DNA column is placed in a collection tube, and the supernatant from the previous step is transferred into the DNA column. The column contains silica that binds to DNA in a high-salt environment. After centrifugation, impurities are removed into the collection tube, which is then discarded. Finally, the DNA column is transferred to a new collection tube for the next step. This process ensures efficient DNA binding while removing contaminants..

[Audio] We add 500 microliters of the buffer to the DNA column and mix it. Then, we centrifuge and discard the collection tube. Since the collection tube contains all the cell impurities and the DNA still bound with silica of the DNA column, we do this twice. We repeat another washing, discard the collection tube again, and after the second washing, we centrifuge the DNA column once more to ensure that we can remove any residual buffer that may be present in the DNA. The collection tube is again discarded, as this step aims to remove any impurities and wash off any contaminants. This procedure must be performed with a high salt concentration to keep the DNA bound to the membrane of the DNA column..

[Audio] The DNA column, which still contains the DNA from the sample, is transferred into a nuclease-free test tube. A buffer with a low salt concentration is added, allowing the DNA to dissociate from the silica membrane. The tube is then incubated and centrifuged, resulting in the collection of the DNA in the nuclease-free test tube. This marks the final step in the silica gel DNA extraction method..

[Audio] The silica gel-based method supports the spin column principle, which means it doesn't require any hazardous chemicals. This method is known for its good quality DNA yield and is widely accepted due to its simplicity. However, each laboratory may have variations in their procedures depending on the type of reagents and chemicals they use, as well as the availability of machines and equipment. Steps may be added, removed, or repeated to improve the procedure, but the basic steps we're discussing are generally applicable across methods..

[Audio] The silica gel method follows the spin column principle and is widely used because it's simple, safe, and provides high-quality DNA without hazardous chemicals. Each laboratory may adjust the procedure based on available reagents, equipment, and specific needs, but the core steps remain the same..

PLANT SAMPLE.

[Audio] The purified RNA is then washed twice with a buffer solution to further improve its purity. The DNA column is mixed with 500 microliters of the buffer, centrifuged, and the collection tube is discarded. This step is repeated twice to remove any remaining impurities and contaminants. After the second washing, the DNA column is centrifuged again to remove any residual buffer. The collection tube is discarded once more, ensuring that the DNA remains bound to the silica gel membrane..

[Audio] This extraction buffer, also known as the lysis buffer, is a solution used to break open cells, dissolving their cellular and nuclear membranes. It plays a crucial role in removing contaminants, ultimately making DNA suitable for molecular biology experiments. By understanding the extraction buffer, we can better comprehend the subsequent steps involved in the process..

[Audio] The composition of the extraction buffer plays a crucial role in breaking down the cell and nuclear membranes, denaturing proteins, removing magnesium ions, maintaining a stable pH, neutralizing DNA's negative charge, and making DNA less hydrophilic. These key components work together to facilitate the purification of DNA, ensuring its integrity and quality for further analysis..

[Audio] Plant cells have a tough cell wall, so we need to modify the protocol to extract DNA. We can do this by using a combination of mechanical, chemical, and enzymatic methods. One way to achieve this is by grinding the plant sample using a mortar and pestle with liquid nitrogen. After grinding, we can then proceed with the digestion by transferring the ground sample to an Eppendorf tube and adding an extraction buffer. The extraction buffer causes the lysis of the cell membrane, breaking it down and releasing the DNA inside the cell..

[Audio] Incubate the plant sample with the extraction buffer at 60 degrees Celsius for 20 to 40 minutes, using a water bath. After the incubation, centrifuge the sample at 10000 rpm for 10 minutes. This helps remove debris, which forms a pellet at the bottom of the tube. Collect the supernatant as the DNA, now referred to as the crude lysate. Transfer the crude lysate into a new Eppendorf tube, discarding the pellet..

[Audio] The mixture has been separated into three distinct layers. The top layer is the aqueous phase, which contains the water-soluble molecules, including our target DNA. Below this, we find the interphase, where plant debris has accumulated. At the bottom of the tube, we have the organic phase, which contains proteins and lipids that have been separated from the rest of the mixture. We carefully collect the aqueous phase in a new tube, taking care not to disturb the lower layers. It's essential to use wide-bore pipette tips to avoid causing mechanical damage to the DNA during this process..

[Audio] After the centrifugation, we will now have a DNA pellet. Purification involves washing the DNA pellet with ethanol. First, we decant the supernatant and add 70% ethanol to wash the DNA pellet. Mixing the ethanol and pellet together removes any residual and contaminants. Next, we centrifuge at 10000 rpm for five minutes. Another DNA pellet appears after centrifugation. We discard the ethanol again, ensuring no residual ethanol remains. After removing the supernatant ethanol, we dry the pellet at room temperature for thirty minutes. Then, we re-suspend the pellet with TE buffer or nuclease-free water. Using highly pure nuclease-free water is crucial, as nucleases like DNase and RNase can degrade the molecular sample if present..

[Audio] The process of extracting Coronavirus RNA using silica gel column is a crucial step in PCR testing. This method allows us to isolate the genetic material from the virus, making it possible to identify its presence. The first step is to collect a sample using a swab, which is then processed through a lysis buffer to break down the cells and release the genetic material. The resulting mixture is then passed through a silica gel column, which separates the DNA from other contaminants. This method has several advantages, including being cheap and easy to perform, but also requires multiple steps and the use of toxic chemicals. Despite this, it remains a widely used technique in molecular biology due to its ability to yield large quantities of high-quality DNA..

Blood Sample.

[Audio] The Proteinase K method is a type of inorganic DNA extraction that breaks down proteins in a blood sample using a Proteinase K solution. This process releases DNA from its complex with proteins, making it suitable for further analysis such as PCR testing. By treating the sample with Proteinase K, we can isolate the DNA and eliminate any interfering substances that may impact the accuracy of our results..

[Audio] The name "Proteinase" originated from the words "protein," referring to long chains of amino acids, and "ase," meaning enzyme. The letter "K" represents Keratin, as the enzyme can break down this tough protein found in hair. This association stems from the fact that Proteinase was the first enzyme isolated from the fungus Tritirachium album, which can digest Keratin. As a result, the "K" suffix was assigned to the enzyme. Additionally, Proteinase K plays a crucial role in DNA extraction, earning it the title of the enzymatic method..

[Audio] In this step, we add a specimen of 2 milliliters of blood to 10 to 20 milliliters of TE buffer, which is a combination of Tris and EDTA. This buffer plays a crucial role in dissolving DNA, breaking down proteins, and helping to lyse the cell and nuclear membranes. After mixing, we centrifuge the solution at 2500 revolutions per minute for 20 minutes. Once the centrifugation is complete, we discard the supernatant, which is the top layer of the liquid. Then, we add a fresh batch of TE buffer, mix it gently, and incubate the solution at a high temperature of around 60 to 65 degrees Celsius for approximately 2 hours. This completes the lysis process..

[Audio] After incubation, we centrifuge the sample at 2,500 rpm for 15 minutes. We then discard the supernatant. Next, we add 20 microliters of Proteinase K solution and 20 milliliters of DNA extraction buffer to the tube. Finally, we incubate the sample at a high temperature of 60-65 degrees Celsius for 2 hours. During this process, the Proteinase K solution breaks down all the proteins present within the cells. The high temperature also helps to denature these proteins. Note that the optimal activity of Proteinase K is recorded at a temperature of 60 degrees Celsius, ranging from 37 to 70 degrees Celsius. Additionally, the enzyme's stability is highest at a pH of 8..

[Audio] Add one to two milliliters of chilled isopropanol and a small amount of sodium chloride to the tube. Invert the tube several times to allow the mixture to settle and form a precipitate. Centrifuge the tube at a speed of eight thousand to ten thousand revolutions per minute to ensure that the precipitate settles at the bottom of the tube. Discard the remaining liquid, also known as the supernatant..

[Audio] We will now purify our DNA further by washing it with ethanol. One millilitre of ethanol will be added to the pellet, followed by spinning it down at 10000 revolutions per minute for approximately one to two minutes. After that, the ethanol will be discarded, and the pellet will be dried. To rehydrate the DNA, we can use a hydration buffer or a TE buffer, and incubate it at a water bath for two to three hours. Alternatively, we can incubate it at room temperature overnight with gentle shaking. This step is crucial in preparing our DNA for PCR testing..

[Audio] The Proteinase K method is a successful DNA extraction method that is the first choice for genomic extraction for microarray and DNA sequencing. It produces highly pure DNA with a good quantity, and the process is simple and rapid. Additionally, it is also used for mRNA extraction. However, the method has a limitation, which is the low shelf-life of Proteinase K, requiring storage at 4 degrees Celsius to maintain its effectiveness..

[Audio] The Proteinase K method is indeed a successful DNA extraction technique, widely used for genomic extraction in applications such as microarray analysis and DNA sequencing. The resulting DNA is highly pure and of good quantity, making it suitable for various downstream applications. Furthermore, the method is simple, rapid, and accurate, and can also be employed for mRNA extraction. However, there is a limitation associated with Proteinase K itself, as it is an enzyme with a limited shelf life. To maintain its potency, it is essential to store it at a controlled temperature, typically around 4 degrees Celsius..

SUMMARY OF PROCEDURES.

[Audio] After the centrifugation, we will mix the DNA-containing aqueous layer with ethanol to purify the DNA. We will then centrifuge the mixture at 1000 rpm for five minutes. Following centrifugation, the DNA pellet will become visible. We will discard the ethanol and dry the pellet at room temperature for thirty minutes. Finally, we will dissolve the pellet in Tris-EDTA buffer..

[Audio] The final step in the process of extracting Coronavirus RNA using silica gel column involves assessing the DNA's concentration and quality. This can be done using a spectrophotometer to measure concentration and purity, or gel electrophoresis to check for DNA presence and quality. Once DNA extraction is complete, the extracted DNA can be used for various downstream applications such as PCR, sequencing, fingerprinting, cloning, and more..