Nucleic Acid Extraction PDF

## Nucleic Acid Extraction ### DNA and RNA DNA is genetic material of human and all living creatures. It is also a protein producer. - **DNA collection** is essential for: - **Molecular Analysis:** including genetic testing, forensic analysis, and cancer research. - **Forensic Analysis:** DNA f...

## Nucleic Acid Extraction ### DNA and RNA DNA is genetic material of human and all living creatures. It is also a protein producer. - **DNA collection** is essential for: - **Molecular Analysis:** including genetic testing, forensic analysis, and cancer research. - **Forensic Analysis:** DNA fingerprinting can be used to identify individuals and match samples in criminal investigations. - **Sensitivity:** DNA is highly sensitive to contamination. - **Forensic Analysis:** DNA can be used to identify individuals, identify relatives, and determine the paternity of individuals. - **Different felid and special for detection:** DNA can be used to identify individuals and match samples in criminal investigations. - **Mitochondrial DNA:** This is a type of DNA found in the mitochondria of cells. It is inherited from the mother. It can be used to trace maternal lineage or to identify individual relatives. - **Cells contain DNA in nucleus and mitochondria:** The nucleus is the control center of the cell, and the mitochondria are the powerhouses of the cell, where energy is produced. These two structures both contain DNA. - **Non coding Part of DNA:** This is the portion of DNA that does not code for proteins. - **Expression of DNA:** This refers to the process of genes being turned on or off. - **Exones coding Port give Protin of DNA & مثال - Bgoblin:** The exons are the parts of DNA that code for protons. The Bglobin (beta globin) is a protein that is responsible for transporting oxygen in the blood. ### DNA Extraction: - The purpose of DNA extraction is to isolate DNA from the cell. This involves several steps: - **Penetration:** The first step is to lyse the cell membrane and nuclear membrane. This is done by using a detergent or enzyme that breaks down the membranes, dissolving the membranes and releasing the cytoplasm. - **Remove cytoplasm:** The next step is to remove the cytoplasm, which contains proteins and other cellular components that can interfere with the DNA analysis. This can be done in many ways including centrifugation, filtration, or precipitation. - **Remove nuclear membrane:** To isolate the DNA that is in the nucleus, the nuclear membrane needs to be destroyed. - **Collection DNA:** This requires proper technique to preserve DNA integrity and minimize contamination. ### Expression of Antibody - **Simple Tantam disease:** This is a disease that is caused by a mutation in a single gene. - **Structure of human cell:** Human cells are made up of many different parts including the cell wall, the cell membrane - **Differences in the intron:** Variation in the DNA sequence of the intron can be used to identify different individuals. Even identical twins will have some small differences in intron sequences because of mutations that occurred during their development. ### DNA extraction (Steps) - **Lysing cell membrane and nuclear membrane:** This is done by using a detergent or enzyme that breaks down the membranes. - **Remove Protein from cytoplasm:** This step occurs after the cell membrane and nuclear membrane have been lysed, allowing for the removal of proteins, which are then removed from the cytoplasm using a detergent or enzyme. - **Collection DNA:** This is the final step in DNA extraction, and the DNA is collected from the remaining solution using techniques like precipitation, centrifugation, or filtration. ## Physical Characteristics of DNA - **Spectrophotometer:** This is a tool used to measure the absorbance of light. It can be used to determine the concentration of DNA. * **Requirements:** * 1 ml of DNA sample * **Method:** * The DNA sample is placed in a spectrophotometer and the absorbance of light is measured. * The absorbance of light is measured at 260 nm. This is the wavelength at which DNA absorbs light maximally. * The concentration of DNA can be calculated using the Beer-Lambert law, which states that the absorbance of a solution is directly proportional to its concentration. - **Nanodrop:** This is a more modern device that can accurately quantify DNA concentration and purity. It is preferred for its small sample size requirement and speed. - **Gel Electrophoresis:** Gel electrophoresis separates DNA fragments based on their size. DNA is then visualized using a fluorescent stain like ethidium bromide. It can be used to determine the size of the DNA fragments. - **Evaluation of DNA:** This refers to the process of assessing the quality and quantity of the DNA sample. - **Water Soluble:** DNA is soluble in water. The DNA can be easily dissolved in water and this can be used to create a solution containing DNA at a specific concentration. - **Viscose:** This refers to DNA's property of being sticky and viscous because it is a long molecule. - **Best Besa Fressing Soluble Cycle:** This refers to a type of DNA extraction that uses a cycle (that involves several steps) to isolate DNA by dissolving it in a solution. - **Precipitates in Alcohols:** DNA can be precipitated out of solution by the use of alcohols like ethanol or isopropanol. - **Tris – HCl:** This buffer is used in DNA extraction. It helps to maintain a constant pH and acts as a protective agent. - **Protein:** Proteins are complex polymers that are present in the cytoplasm and can interfere with DNA analysis. They are removed to purify the DNA. - **Collection Buffer:** This buffer is used to collect the DNA after it has been precipitated. - **Carry-ve change:** DNA is negatively charged, meaning that it can be separated by gel electrophoresis. Since it has a negative charge it moves towards the positive electrode during electrophoresis. - **Melting and Annealing Temperature of PCR:** This refers to the specific temperatures used during the Polymerase Chain Reaction (PCR) technique. - **PCR (Polymerase Chain Reaction):** This method is used to amplify DNA and is important for many DNA analysis techniques. ## Purity of DNA - **Protein:** The DNA sample should be free of any protein contamination. This can be determined using assays such as the Bradford assay. - **Hyparintube:** This is a type of tube used for DNA analysis. It has a special coating that helps to prevent DNA degradation. - **Concentration of DNA:** The DNA concentration in the sample should be high enough for further analysis. - **Ethanolamine and isopropanol:** These are substances that can interfere with the DNA analysis. They should be avoided. - **Concentration of DNA:** This should be in the range of 10 ng-10 μl. ## Electrophoresis - **Pre-PCR:** This set of steps is used to evaluate DNA prior to amplification by Polymerase Chain Reaction (PCR). - **Pre-PCR DNA Evaluation:** This is done to determine the quality and quantity of the DNA sample. - **Analysis Using Pre-PCR:** This is done to ensure that the DNA is suitable for PCR amplification. - **Post-PCR:** This is done after the PCR reaction has been completed. - **Post-PCR Analysis:** This is done to evaluate the PCR products and to ensure that they are of the correct size and quality. ## Reasons For Doing DNA Extraction - **Disease Diagnosis:** This involves searching for genetic abnormalities that can cause disease (diagnostic testing). - **DNA Sequencing:** This is a method used to determine the order of nucleotides (base sequences) in DNA, providing genetic information of any organism. ### DNA Sequencing: - **Determining the order of nucleotides:** The order of the nucleotide bases, A, T, C and G, in a section of DNA is determined using techniques like Sanger sequencing. This information is essential for understanding the function of genes and for identifying mutations and genetic variations. - **Genetically modified organism (GMO):** This is an organism whose genetic makeup has been altered by the introduction of genes from another organism. This can be used to improve the quality of crops, create new medicines, or provide insights into fundamental biological processes. ## Sample Source - **Blood:** This is an important source of DNA, with both white blood cells (WBCs) and red blood cells (RBCs). - **Whole blood:** This contains both WBCs and RBCs. Only the WBCs contain DNA because RBCs lack nuclei. Therefore, RBCs need to be removed. - **Red Blood Cell Lysis Buffer (RCLB):** This buffer is used to lyse RBCs, a key step in isolating WBCs and their DNA. - **Centrifuge (santer fuge):** This is a vital tool for separating components of a sample, such as RBCs from WBCs, due to their different densities. - **Buffy Coat:** This layer between RBCs and plasma-rich serum in a centrifuged blood sample contains WBCs, which is a rich source of DNA for extraction. - **Ficoll:** This is a density gradient medium used in centrifugation to isolate WBCs from blood. - **Blood Sample:** This is a valuable source of DNA for various applications like paternity testing, forensic analysis, and medical diagnosis. - **High Resh of cell:** This refers to a high concentration of cells, making a sample more suitable for DNA extraction. ### Different Types of Samples - **Plasma:** Plasma is the liquid part of blood after removal of blood cells. It is rich in proteins, hormones, and other factors but does not contain nuclei, rendering it unsuitable for DNA extraction. - **Dry blood spot:** This is a sample of blood that has been dried onto filter paper. This method is often used for newborn screening and for collecting samples in remote areas. - **Buccal cell:** These cells are collected by scraping the inside of the cheek, and contains DNA that can be analyzed for a variety of applications such as identifying individuals. It is a non-invasive source of DNA. ## Buccal Cell Processing - **Buccal cell PBS:** This is a buffer solution used to collect buccal cells for DNA extraction. - **Phosphate-buffered saline (PBS):** This is a liquid solution used to collect cells for DNA extraction. It is a physiological solution and also keeps cell function stable. ## DNA Extraction Methods: - **Preservation of cells:** This refers to methods that maintain cell integrity and viability, which are crucial for successful DNA extraction. - **Normal saline:** This refers to a solution that is similar in salt concentration to the human body. It can be used to wash and preserve cells. - **Three important factors:** 1. **Processing speed:** The time required for sample processing. Faster methods are generally preferred. 2. **Ease of Use:** The complexity of the procedures involved. Simple, user-friendly methods are desirable. 3. **Cost of preparation:** The cost of reagents, equipment, and lab consumables involved in the extraction process. ### DNA Extraction Method Generations: - **First Generation:** This method is very old, taking 3 days for completion. - **Second Generation:** This generation is more efficient, taking 1 hour to complete. - **Third Generation:** This generation is much faster than the previous methods, taking only 30 minutes, but it is also much more expensive. - **Fourth Generation:** The most modern generation takes only 5 minutes, but it is also much more expensive than the previous methods. - **ICU:** This is a critical care unit or intensive care unit. ## Storage of Sample - **DNA and RNA:** These are sensitive molecules that degrade quickly under certain conditions. Therefore, careful storage is important. - **22-25 degrees Celsius:** Not recommended for longer storage periods. The DNA and RNA can degrade quickly at this temperature. - **2 to 8 degrees Celsius:** This temperature range is used for short-term storage, up to 72 hours. - **-20 degrees Celsius:** This temperature range is used for mid-term storage, up to several weeks. - **-70 degrees Celsius:** The preferred storage temperature for long-term storage, up to several years. - **Blood and Bone marrow:** These samples should not be frozen. - **Four forms of Blood storage:** 1. **Buffy Coat:** This is the layer of white blood cells that is separated from the rest of the blood by centrifugation. 2. **Serum:** Serum is the liquid portion of blood that remains after clotting. 3. **Plasma:** This is the liquid part of blood that remains after the blood cells have been removed. Plasma contains lots of factors that can affect DNA and RNA quality over time. 4. **Dry blood spot:** This method of storage involves applying a small drop of blood to filter paper and letting it air dry. ## RNA Storage - **RNA:** RNA is a much less stable molecule than DNA. It can be degraded quickly by enzymes called RNAses. RNA degradation can occur due to exposure to heat, oxygen, or other factors. - **22-25 degrees Celsius:** Not recommended for longer storage period. RNA is very sensitive to breakdown at room temperature. - **2 to 8 degrees Celsius:** This temperature range is used for short-term storage, up to 2 hours. - **-20 degrees Celsius:** This temperature range is used for mid-term storage, up to 24 hours. - **-80 degrees Celsius:** This temperature range is used for long-term storage, up to 2-4 weeks. - **Do not freeze blood or bone marrow before lysing red blood cells (RBCs ):** Cell lysis is a necessary step to prevent degradation of RNA. ## DNA Extraction (Lysing cells) - **Cell wall lysis:** To extract DNA, the cell wall needs to be disrupted using a buffer solution. - **Lysis buffer:** This is a solution that contains detergents and enzymes that break down the cell wall and membranes, allowing for the release of DNA. - **Precipition of proteins:** This step removes proteins from the DNA sample. - **Precipition of DNA:** This step isolates the DNA from the rest of the cell components. It involves using ethanol or isopropanol. - **Washing:** This process removes residual contaminants from the DNA to ensure purity. - **Tris-EDTA Buffer:** Tris-EDTA is a buffer frequently used to keep DNA stable and minimize DNA degradation during extraction.