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课程主页: https://www.udemy.com/course/dna-extraction-learn-to-extract-dna/
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**DNA提取：掌握DNA提取技术** 本课程将深入探讨DNA提取的全过程，一种通过物理或化学方法从样本中纯化DNA的技术。我们将追溯DNA分离的起源，了解弗里德里希·米歇尔在1869年首次进行的DNA分离。 课程将重点关注高效DNA提取的关键要素：获取高产量、高质量且纯净的DNA，同时要确保去除RNA和蛋白质等污染物。我们将介绍手动方法以及市售试剂盒的应用。 您将学习如何从各种样本中提取DNA，包括血液、体液，细针穿刺抽吸细胞学（FNAC）标本，福尔马林固定石蜡包埋（FFPE）组织，以及冷冻组织切片等。 **课程内容亮点：** * **DNA提取的核心步骤：** 细胞裂解、DNA溶解，以及用于去除大分子、脂质、RNA和蛋白质的化学或酶法处理。 * **经典提取技术：** * **有机提取（酚-氯仿法）：** 详细解析这一耗时但经典的实验方法，包括细胞裂解、蛋白质变性、有机溶剂处理、离心分离和RNA酶处理。 * **非有机法：** 探讨盐析法和蛋白酶K处理等方法。 * **吸附法：** 介绍硅胶膜技术。 * **其他先进技术：** 了解硅胶基技术、磁分离技术、阴离子交换技术、盐析法以及氯化铯密度梯度离心法。 * **DNA质量与产量评估：** 学习使用分光光度法（重点关注A260/A280比值）和凝胶电泳来评估DNA的纯度和浓度。 通过本课程，您将全面掌握DNA提取的原理、方法和评估技术，为您的科研或实际应用打下坚实基础。
DNA extraction is a method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components. Friedrich Miescher in 1869 did DNA isolation for the first time.The use of DNA isolation technique should lead to efficient extraction with good quantity and quality of DNA, which is pure and is devoid of contaminants, such as RNA and proteins. Manual methods as well as commercially available kits are used for DNA extraction. Various tissues including blood, body fluids, direct Fine needle aspiration cytology (FNAC) aspirate, formalin-fixed paraffin-embedded tissues, frozen tissue section, etc., can be used for DNA extraction.DNA extraction involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove macromolecules, lipids, RNA, or proteins.DNA extraction techniques include organic extraction (phenol-chloroform method), nonorganic method (salting out and proteinase K treatment), and adsorption method (silica-gel membrane).Organic extraction:This method is labor intensive and time consuming.Cell lysis can be done using nonionic detergent (sodium dodecyl sulfate), Tris-Cl, and Ethylene diamine tetraacetic acid (EDTA), and this step is followed by removal of cell debris by centrifugation. Protease treatment is then used to denature proteins. Organic solvents such as chloroform, phenol, or a mixture of phenol and chloroform (phenol/chloroform/isoamyl alcohol ratio is 25:24:1) are used for denaturation and precipitation of proteins from nucleic acid solution, and denatured proteins are removed by centrifugation and wash steps. RNAse treatment is done for the removal of unwanted RNA. Precipitation with ice-cold ethanol is performed for concentrating DNA. Nucleic acid precipitate is formed, when there is moderate concentration of monovalent cations (salt). This precipitate can be recovered by centrifugation and is redissolved in TE buffer or double-distilled water.Other methods include silica-based technology (DNA absorbs to silica beads/particles at a specific pH in presence of specific salts), magnetic separation (DNA binds reversibly to magnetic beads, which are coated with DNA-binding antibody), anion exchange technology, salting out, and cesium chloride density gradients.Assessing the quality and yield of DNA: The quality and yield of DNA are assessed by spectrophotometry or by gel electrophoresis. Spectrophotometry involves estimation of the DNA concentration by measuring the amount of light absorbed by the sample at specific wavelengths. Absorption peak for nucleic acids is at ~260 nm. The A260/A280 ratio is ~1.8 for dsDNA. A ration of less than 1.7 indicates protein contamination.
